Mdr e. coli immunogen

ABSTRACT

The subject relates to an isolated antibody that specifically binds to O25b antigen of multi drug resistant (MDR) E. coli strains, its medical and diagnostic use, method of producing the antibody, including an isolated nucleotide sequence, plasmids and host cells as used in the production of the antibody; and further an isolated epitope recognized the specific antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Pat. Application No.17/167,595, filed Feb. 4, 2021 (now allowed), which is a divisionalapplication of U.S. Pat. Application No. 16/229,223, filed Dec. 21,2018, now U.S. Pat. US 10,940,191 B2, issued Mar. 9, 2021, which is acontinuation application of U.S. Pat. Application No. 15/373,528, filedDec. 9, 2016, now U.S. Pat. US 10,206,992, issued Feb. 19, 2019, whichis a divisional application of U.S. Pat. Application No. 14/651,156,filed Jun. 10, 2015, now U.S. Pat. US 9,849,169 B2, issued Dec. 26,2017, which is a national stage application of International ApplicationNo. PCT/EP2014/050895, filed Jan. 17, 2014, which claims priority toEuropean Patent Application No. 13151627.0, filed Jan. 17, 2013. Theentire contents of the above-identified applications are hereby fullyincorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing named “004852-197US5Sequence Listing” (size 421 KB) with a creation date of Nov. 22, 2022,is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention refers to an antibody specifically binding to the LPS O25bantigen of multi drug resistant (MDR) E. coli strains.

BACKGROUND OF THE INVENTION

Lipopolysaccharide (LPS) is the most abundant antigen on the surface ofenterobacterial pathogens. Typically, LPS has three structural parts: i)Lipid A (also known as endotoxin), ii) core oligosaccharide, and iii)O-antigen. The latter is made up of repeating subunits of 3-6 sugars(depending on the serotype). Lipid A and core OS are relatively wellconserved in one single enterobacterial species, however, theiraccessibility to antibodies are limited. On the other hand, O-antigensare highly accessible, but very diverse with respect to their structure(in E. coli there are ~180 different O-types).

Antibodies against O-antigens are able to bind to the surface of E.coli, hence they are used both for diagnostics (e.g., O-typing forepidemiology studies) as well as are proposed as therapeutic measures.Nevertheless, given the huge structural variability, a broad-spectrumprotection with O-antigen specific antibodies is cumbersome.

Extraintestinal infections caused by E. coli are common causingsignificant morbidity and mortality. Multi-drug resistant (MDR) strainsof E. coli that have emerged recently cause a significant proportion ofE. coli infections.

Treatment options against these MDR strains are getting very limited asthey have evolved resistance to most classes of clinically relevantantibiotics. Therefore, an alternative treatment option, e.g., passiveimmunization with monoclonal antibodies (mAbs) holds a great promise forthe future.

In the past years a well-defined clonal lineage of MDR E. coli. theST131-O25b:H4 has emerged causing approximately 10% of allextraintestinal E. coli infections and about half of the MDR E. coliinfections (Peirano et al. Int J Antimicrob Agents 2010Apr;35(4):316-21; Rogers et al. J Antimicrob Chemother 2011Jan;66(1):1-14; Woodford et al. FEMS Microbiol Rev 2011Sep;35(5):736-55). Strains belonging to this lineage show limitedheterogeneity thus could be considered very similar with respect toantigenic repertoire. The vast majority of the strains belonging to thiscluster express the O25b antigen and hence a specific gene (within theLPS synthesis locus) encoding for enzymes synthesizing this antigen isused for the identification of this clone (Clermont et al. J AntimicrobChemother 2008 May; 61(5):1024-8.). Alternatively, agglutination withthe O25 typing sera can be used, in spite that the O antigen of thislineage differ from the classical O25 antigen (hence it had been termedO25b) as suggested by genetic differences. However, the O25 typing seracannot distinguish between the non-MDR O25 and the MDR O25b clones.

Rogers et al. (J Antimicrob Chemother 2011 Jan;66(1):1-14) describe thedetection of the E. coli O25b-ST131 strain by three majorcharacteristics, i.e., its serogroup (O25b), its phylogenetic group (B2)and its ST (ST131). Each of these characteristics is disclosed to aiddetection. A variety of molecular techniques is described, i.e. MLST,PCR-based rapid detection methods, repetitive sequence PCR and PFGE.Polyclonal antisera (raised against an O25a strain) including a varietyof immunoglobulins have been used to determine the O25 antigen, notdifferentiating subtypes.

Jadhav et al. (PLOS ONE 2011;6(3): e18063) describe the virulencecharacteristics and genetic affinities of strains which were positivefor the O25b subgroup that is linked to the B2-O25b-ST131-CTX-M-15virulent/ multi-resistant type. Human clinical isolates were analyzedand classified into serotypes and virulence marker profiles wereobtained. O25 positive strains were identified by serotyping usingpolyclonal antisera against O-antigens – O1 to O173. The O25 positivestrains were further subjected to genotyping by an allele-specific PCRtargeting the rfbO25b subgroup gene locus.

Mora et al. (Int. J. Antimicrobial Agents 2011;37(1): 16-21) describethe emergence of some clonal groups among CTX-M-14 producing E. coliclinical isolates, among them O25b: H4-B2-ST131. O typing was done withspecific O antisera (polyclonal).

Clermont et al. (J Antimicrob Chemother 2008 May; 61(5):1024-8)discloses an allele-specific pabB PCR assay specific for O25b ST131 E.coli.

Szijarto et al, (FEMS Microbiol Lett 2012;332:131-6) describe moleculartyping of E. coli strain isolates based on the core structure of the LPSmolecule. The core type of the isolates was determined by PCR usingprimers targeting genes in the core operon and specific to R1-4 and K-12core types, respectively.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide for an antibodydirected against MDR strains of E. coli with improved specificity to beused for the prevention or therapy of E. coli infections caused by LPSO25b carrying strains. It is further the objective to provide means andmethods that are capable of diagnosing MDR E. coli bacteria in a rapidand reliable manner.

The object is solved by the subject of the present invention.

According to the invention there is provided an isolated antibody thatspecifically binds to O25b antigen of multi drug resistant (MDR) E. colistrains.

Specifically, the antibody is a monoclonal antibody.

Specifically, the antibody is specific to bind O25b antigen only, orcross-specific to bind an epitope shared by the O25a and O25b antigens.

According to a specific aspect, the antibody is cross-specific to bindthe O25b and O25 or O25a antigens, e.g., with equal, more than equal,similar or different affinities.

Specifically, the antibody of the invention preferentially binds to theO25b antigen relative to the O25a antigen of E. coli, or at least withequal affinity towards both antigens.

According to a specific embodiment, the antibody has at least two-foldgreater affinity for binding the O25b antigen as compared to the O25aantigen, specifically with at least two-fold difference, or at leastthree-fold, at least four-fold, at least 5-fold, or even at least10-fold difference in binding either the O25b or the O25a antigen, e.g.,difference in affinity and/or avidity.

According to a specific aspect the specific binding to O25b ischaracterized by the greater affinity for binding the O25b antigen ascompared to binding the O25b antigen by a polyclonal serum raisedagainst O25 or O25a E. coli strains as determined by immunoassay,preferably immunoblotting, ELISA or other immunological methods. Thehigher binding affinity is specifically with at least two-folddifference, or at least three-fold, at least four-fold, at least 5-fold,or even at least 10-fold difference.

Specifically, the O25b antigen as targeted by the antibody of theinvention is prevalent in one or more, and more specifically present inthe vast majority of ST131 strains.

Specifically, the epitope recognized by the antibody is present on thesurface of encapsulated and non-encapsulated ST131-O25b:H4 strains,e.g., mutant strains.

According to a further specific aspect, the antibody has a binding siteof a full-length monoclonal antibody or an antibody fragment thereofcomprising at least one antibody domain incorporating a binding site,which antibody is preferably an antibody selected from the groupconsisting of murine, lama, rabbit, goat, cow, chimeric, humanized orhuman antibodies, heavy-chain antibodies, Fab, Fd, scFv andsingle-domain antibodies like VH, VHH or VL, preferably a human IgGantibody or a murine IgG antibody.

According to a further specific aspect, the antibody has an affinity tobind the O25b antigen with a Kd of less than 10⁻⁷ M, preferably lessthan 10⁻⁸ M, specifically in a monomeric state.

According to a further specific aspect, the antibody exhibits in vitrobactericidal potency in a serum sample comprising live wild-type MDR E.coli strains.

According to a further specific aspect, the antibody stimulates uptakeof live wild-type MDR E. coli strains by phagocytic cells in vitro.

According to a further specific aspect, the antibody binds the sameepitope as the antibody designated as 8D5-1G10 or 8D10-C8.

According to a further specific aspect, the antibody comprises the samebinding site as the antibody designated as 8D5-1G10 or 8D10-C8.

According to a specific aspect, the invention provides for an isolatedmonoclonal antibody that specifically binds to O25b antigen of multidrug resistant (MDR) E. coli strains which comprises the antigen-bindingsite of antibody 8D5-1G10, or which is derived from the antibody8D5-1G10, or a functionally active variant of the antibody 8D5-1G10,preferably wherein the antibody 8D5-1G10 is characterized by

-   a) the variable region of the antibody light chain produced by the    host cell deposited under DSM 26763; and/or-   b) the variable region of the antibody heavy chain produced by the    host cell deposited under DSM 26762;-   c) or a functionally active variant of (a) and/or (b) is employed.

According to a specific embodiment, the antibody is the 8D5-1G10antibody, or a functionally active variant thereof.

Further antibodies of the invention are exemplified herein, which aredesignated 6D1-1B2 and 8A1-1G8. These are clones with CDR sequencessimilar to the 8D5-1G10, herein also understood as functionally activeCDR variants.

Specifically, the antibody designated as 8D5-1G10 is composed of anantibody light chain comprising the variable region encoded by thecoding sequence of the plasmid comprised in the E. coli host celldeposited under DSM 26763, and an antibody heavy chain comprising thevariable region encoded by the coding sequence of the plasmid comprisedin the E. coli host cell deposited under DSM 26762.

According to a further specific aspect, the antibody is derived from the8D5-1G10 antibody, wherein

-   the variable region of the antibody light chain is encoded by a    plasmid comprised in the E. coli host cell deposited under DSM    26763, or a functionally active variant thereof; and/or-   the variable region of the antibody heavy chain is encoded by a    plasmid comprised in the E. coli host cell deposited under DSM    26762, or a functionally active variant thereof.

According to a further specific aspect, the antibody is derived from anantibody, wherein

-   the variable region of the antibody light chain is produced by a    host cell deposited under DSM 26763, or a functionally active    variant thereof; and/or-   the variable region of the antibody heavy chain is produced by a    host cell deposited under DSM 26762, or a functionally active    variant thereof.

According to another specific aspect, the invention provides for anisolated monoclonal antibody which is cross-specific to bind an epitopeshared by the O25a and O25b antigens, and which comprises theantigen-binding site of antibody 8D10-C8, or which is derived from theantibody 8D10-C8, or a functionally active variant of the antibody8D10-C8, preferably wherein the antibody 8D10-C8 is characterized by

-   a) the variable region of the antibody light chain produced by the    host cell deposited under DSM 28171; and/or-   b) the variable region of the antibody heavy chain produced by the    host cell deposited under DSM 28172;-   c) or a functionally active variant of (a) and/or (b) is employed.

According to a specific embodiment, the antibody is the 8D10-C8antibody, or a functionally active variant thereof.

Specifically, the antibody designated as 8D10-C8 is composed of anantibody light chain comprising the variable region encoded by thecoding sequence of the plasmid comprised in the E. coli host celldeposited under DSM 28171, and an antibody heavy chain comprising thevariable region encoded by the coding sequence of the plasmid comprisedin the E. coli host cell deposited under DSM 28172.

According to a further specific aspect, the antibody is derived from the8D10-C8 antibody, wherein

-   the variable region of the antibody light chain is encoded by a    plasmid comprised in the E. coli host cell deposited under DSM    28171, or a functionally active variant thereof; and/or-   the variable region of the antibody heavy chain is encoded by a    plasmid comprised in the E. coli host cell deposited under DSM    28172, or a functionally active variant thereof.

According to a further specific aspect, the antibody is derived from anantibody, wherein

-   the variable region of the antibody light chain is produced by a    host cell deposited under DSM 28171, or a functionally active    variant thereof; and/or-   the variable region of the antibody heavy chain is produced by a    host cell deposited under DSM 28172, or a functionally active    variant thereof.

Specifically, the functionally active variant is a CDR variant, e.g.which comprises a CDR, more specifically a CDR loop sequence, with anamino acid sequence having at least 60% sequence identity, preferably atleast 70%, 80% or 90% sequence identity.

Specifically, the antibody is derived from such antibodies, employingthe respective CDR sequences, or CDR mutants, including functionallyactive CDR variants, e.g. with 1, 2 or 3 point mutations within one CDRloop.

Specifically, the functionally active variant differs from the parentantibody in at least one point mutation in the amino acid sequence,preferably in the CDR, wherein the number of point mutations in each ofthe CDR amino acid sequences is either 0, 1, 2 or 3.

According to a further specific aspect, the invention provides for aplasmid comprising a nucleotide sequence A

-   encoding the variable region of the antibody light chain designated    8D5-1G10-LC comprised in a host cell deposited under DSM 26763;    and/or-   encoding the variable region of the antibody heavy chain designated    8D5-1G10-HC comprised in a host cell deposited under DSM 26762;

or B

-   encoding the variable region of the antibody light chain designated    8D10-C8-LC comprised in a host cell deposited under DSM 28171;    and/or-   encoding the variable region of the antibody heavy chain designated    8D10-C8-HC comprised in a host cell deposited under DSM 28172.

According to a further specific aspect, the invention provides for anexpression cassette comprising a coding sequence to express a lightchain and/or heavy chain of an antibody of the invention, whichexpression cassette or coding sequence is derived from a plasmidselected from the group consisting of a plasmid of the invention.

According to a further specific aspect, the invention provides for amethod of producing an antibody of the invention, wherein a host cell istransformed with a plasmid of the invention or the expression cassetteof the invention.

According to a further specific aspect, the invention provides for ahost cell comprising a plasmid of the invention or the expressioncassette of the invention.

Specifically, the host cell is deposited under

-   A DSM 26763 and/or DSM 26762;-   or B DSM 28171 and/or DSM 28172.

A specific embodiment refers to a method of producing an antibody of theinvention, wherein a host cell of the invention is cultivated ormaintained under conditions to produce said antibody.

According to a further specific aspect, the invention provides for amethod of identifying a candidate antibody comprising:

-   a) providing a sample containing an antibody or antibody-producing    cell; and-   b) assessing for binding of an antibody in or produced by the sample    with an epitope recognized by the antibody designated as 8D5-1G10 or    8D10-C8, wherein a positive reaction between the antibody and the    epitope identifies the antibody as candidate antibody.

According to a further specific aspect, the invention provides for amethod of identifying a candidate antibody comprising:

-   a) providing a sample containing an antibody or antibody-producing    cell; and-   b) assessing for binding of an antibody in or produced by the sample    with O25b antigen of an ST131-O25b:H4 strain and O25 antigen of a    non-MDR E. coli strain, or the O25a antigen,

wherein a specific positive reaction between the antibody and the O25bantigen relative to the O25 antigen or O25a antigen identifies theantibody as candidate antibody.

Specifically, the candidate antibody is a candidate protective antibody,such as for therapeutic use, or a candidate diagnostic antibody.

Yet, according to a further specific aspect, the invention provides fora method of producing an antibody of the invention, comprising

-   a) providing a candidate antibody identified according to the    invention; and-   b) producing a monoclonal antibody, or a humanized or human form of    the candidate antibody, or a derivative thereof with the same    epitope binding specificity as the candidate antibody.

According to another specific aspect, the invention provides for amethod of producing an antibody of the invention, comprising

-   a) immunizing a non-human animal with an epitope recognized by the    antibody designated as 8D5-1G10 or 8D10-C8;-   b) forming immortalized cell lines from the isolated B-cells;-   c) screening the cell lines obtained in b) to identify a cell line    producing a monoclonal antibody that binds to the epitope; and-   d) producing the monoclonal antibody, or a humanized or human form    of the antibody, or a derivative thereof with the same epitope    binding specificity as the monoclonal antibody.

According to another specific aspect, the invention provides for amethod of producing an antibody of the invention, comprising

-   a) immunizing a non-human animal with O25b antigen of a    ST131-O25b:H4 strain and isolating B-cells producing antibodies;-   b) forming immortalized cell lines from the isolated B-cells;-   c) screening the cell lines to identify a cell line producing a    monoclonal antibody that preferentially binds to the O25b antigen    relative to the O25 antigen or O25a antigen of E. coli; and-   d) producing the monoclonal antibody, or a humanized or human form    of the antibody, or a derivative thereof with the same epitope    binding specificity as the monoclonal antibody.

According to a further aspect, the invention provides for the medicaluse of an antibody of the invention. Specifically, the antibody isprovided for use in treating a subject at risk of or suffering from aMDR E. coli infection comprising administering to the subject aneffective amount of the antibody to limit the infection in the subjector to ameliorate a disease condition resulting from said infection,preferably for treatment or prophylaxis of pyelonephritis, secondarybacteremia, sepsis, peritonitis, meningitis, and ventilator-associatedpneumonia.

Specifically, the antibody is provided for bactericidal killing of MDRE. coli, preferably a ST131-O25b:H4 strain irrespective of capsularpolysaccharide expressed by the strain.

According to a specific aspect, there is further provided a method oftreatment wherein a subject at risk of or suffering from a MDR E. coliinfection is treated, which method comprises administering to thesubject an effective amount of the antibody to limit the infection inthe subject or to ameliorate a disease condition resulting from saidinfection, preferably a method for treatment or prophylaxis ofpyelonephritis, secondary bacteremia, sepsis, peritonitis, meningitis,and ventilator-associated pneumonia.

Specifically, the method of treatment is provided for bactericidalkilling of MDR E. coli, preferably a ST131-O25b:H4 strain irrespectiveof capsular polysaccharide expressed by the strain.

According to a specific aspect, immunotherapy using the antibody of theinvention may effectively protect against live bacterial challenge,e.g., as determined in various animal models.

The antibody may specifically neutralize lethal endotoxaemia. Suchfunctional activity may be determined in an appropriate in vivo model(challenge with purified LPS).

The antibody is specifically effective against MDR E. coli bycomplement-mediated killing, e.g., as determined by an in vitro serumbactericidal assay (SBA), e.g., with at least 20% killing of bacteriaabove the control samples (no antibody or irrelevant control mAb added).

The antibody is specifically effective against MDR E. coli by antibodymediated phagocytosis, e.g., as determined by an in vitroopsonophagocytotic killing assay (OPK), e.g., with at least 20% uptakeof input bacteria or 20% lower end cfu count above the control samples(no antibody or irrelevant control mAb added).

The antibody is specifically effective against MDR E. coli byneutralizing endotoxin functions, e.g., as determined by an in vitro LALassay, or toll-like receptor 4 (TLR4) reporter assay e.g., with at least20% reduction in endotoxin activitiesin comparison to control samples(no antibody or irrelevant control mAb added).

According to a specific embodiment, the antibody is administered in aparenteral or mucosal formulation.

According to a further aspect, the invention provides for apharmaceutical preparation of an antibody of the invention, preferablycomprising a parenteral or mucosal formulation, optionally containing apharmaceutically acceptable carrier or excipient.

According to a further aspect, the invention provides for an antibody ofthe invention, for diagnostic use to detect or determine E. coliinfection in a subject caused by MDR strains expressing the LPS O25bantigen, such as with upper and lower urinary tract infections,including cystitis or urethritis, ascending or hematogenouspyelonephritis, especially in diabetic patients, as well as withbacteremia, sepsis, peritonitis, or intestinal colonization.

Specifically, the antibody is provided for use according to theinvention, wherein a systemic infection with MDR E. coli in a subject isdetermined ex vivo by contacting a sample of body fluid of said subjectwith the antibody, wherein a specific immune reaction of the antibodydetermines the infection.

Specifically, a sample of body fluid is tested for the specific immunereaction, which sample is selected from the group consisting of urine,blood, blood isolates or blood culture, aspirate, sputum, lavage fluidof intubated subjects and stool.

Specifically, the diagnostic use according to the invention refers todetermining the serotype of E. coli in vitro from a pure E. coli culturerecovered from a clinical specimen.

According to a further aspect, the invention provides for a diagnosticpreparation of an antibody of the invention, optionally containing theantibody with a label and/or a further diagnostic reagent with a label,such as a reagent specifically recognizing the antibody or an immunecomplex of the antibody with the respective target antigen, and/or asolid phase to immobilize at least one of the antibody and thediagnostic reagent. The diagnostic preparation may be provided as acomposition or as a kit of parts, e.g. comprising components, such ascomponents comprising

-   a) the diagnostic antibody preparation, and/or-   b) the further diagnostic reagent,-   and/or a solid phase to immobilize at least one of the antibody and    the diagnostic reagent, either as a separate component or as a    carrier of any of components a) and/or b) above.

Preferred diagnostic assays of the invention comprise the antibody ofthe invention immobilized on a solid phase, e.g., latex beads, goldparticles, etc., e.g., to test agglutination by the antibody of bacteriaexpressing O25b antigen or free (or isolated) O25b antigen obtained froma sample to be tested.

Some diagnostic assays may involve two different antibodies withdifferent specificity and/or affinity to bind O25b and/or O25a, so topossibly differentiate between the O25b and O25a antigens.

According to a specific aspect, the invention provides for companiondiagnostics to determine the infection of a subject with MDR E. coli bythe diagnostics of the invention or the diagnostic method of theinvention, to provide for the basis of treatment with a therapeuticagainst such infection, e.g., employing immunotherapy, such as treatingwith an antibody of the invention.

According to a specific aspect, the invention provides for a sensitivebedside diagnostics to diagnose infection of a subject with MDR E. coliby determining free LPS, e.g. from clinical specimen where the amount oflive bacteria is limited. The sensitivity of such assay is specificallyless than 100 ng preferably less than 10 ng of LPS.

According to a further aspect, the invention provides for an isolatedepitope recognized by the antibody designated 8D5-1G10 or 8D10-C8. Suchepitope may consist of a single epitope or a mixture of epitopescomprising epitope variants, each recognized by the specific antibodydesignated 8D5-1G10 or 8D10-C8. Specifically, the epitope of the 8D10-C8antibody is a shared one and prevalent on both the O25b and O25aantigens, thus, the antibody is considered as being cross-specific, yetpreferentially binding the O25b antigen at least with equal as bindingthe O25a antigen.

According to a further aspect, the invention provides for an immunogencomprising:

-   a) an epitope of the invention;-   b) optionally further epitopes not natively associated with said    epitope of (a); and-   c) a carrier.

Specifically, the carrier is a pharmaceutically acceptable carrier,preferably comprising buffer and/or adjuvant substances.

The immunogen of the invention is preferably provided in a vaccineformulation, preferably for parenteral use.

Specifically, the immunogen of the invention is provided for medicaluse, specifically for use in treating a subject by administering aneffective amount of said immunogen to protect the subject from an MDR E.coli infection, or to prevent a disease condition resulting from saidinfection.

Specifically, the immunogen of the invention is provided for eliciting aprotective immune response.

According to a specific aspect, there is further provided a method oftreatment wherein a subject at risk of an MDR E. coli infection istreated, which method comprises administering to the subject aneffective amount of the immunogen to prevent infection in the subject,in particular to protect against pathogenic MDR E. coli.

According to a further aspect, the invention provides for an isolatednucleic acid encoding an antibody of the invention, or encoding anepitope of the invention.

FIGURES

FIG. 1 : Surface staining of different ST131:O25b strains expressingO25b, O25a, or O2 antigens by O25b-specific (with or withoutcross-reactivity to O25a antigen) mAbs.

FIG. 2 : reactivity of different mAbs and O25 rabbit serum to purifiedO25a and O25b LPS molecules in an immunoblot assay.

FIG. 3 : (a): Structure of the repeating unit of E. coli O25b antigen.(b): structure of the repeating unit of E. coli O25a (also referred toO25, sometimes referred to as O25(a)) for comparison (Kenne et al,1985).

FIG. 4 : Detection of O25b antigen expressing E. coli strains withagglutination assay using mAb 8D5-1G10 coupled to latex beads.

FIG. 5 : Detection of soluble O25b antigen with agglutination assayusing mAb 8D5-1G10 coupled to latex beads.

FIG. 6 : Protection provided by passive immunization of mice withO25b-specific murine mAbs against a subsequent intravenous lethalchallenge by an ST131:O25b clinical isolate.

FIG. 7 : Complement dependent bacterial killing mediated by the O25bspecific mAbs.

DETAILED DESCRIPTION

The term “antibody” as used herein shall refer to polypeptides orproteins that consist of or comprise antibody domains, which areunderstood as constant and/or variable domains of the heavy and/or lightchains of immunoglobulins, with or without a linker sequence.Polypeptides are understood as antibody domains, if comprising abeta-barrel structure consisting of at least two beta-strands of anantibody domain structure connected by a loop sequence. Antibody domainsmay be of native structure or modified by mutagenesis or derivatization,e.g., to modify the antigen binding properties or any other property,such as stability or functional properties, such as binding to the Fcreceptors FcRn and/or Fcgamma receptor.

The antibody as used herein has a specific binding site to bind one ormore antigens or one or more epitopes of such antigens, specificallycomprising a CDR binding site of a single variable antibody domain, suchas VH, VL or VHH, or a binding site of pairs of variable antibodydomains, such as a VL/VH pair, an antibody comprising a VL/VH domainpair and constant antibody domains, such as Fab, F(ab′), (Fab)₂, scFv,Fv, or a full length antibody.

The term “antibody” as used herein shall particularly refer to antibodyformats comprising or consisting of single variable antibody domain,such as VH, VL or VHH, or combinations of variable and/or constantantibody domains with or without a linking sequence or hinge region,including pairs of variable antibody domains, such as a VL/VH pair, anantibody comprising or consisting of a VL/VH domain pair and constantantibody domains, such as heavy-chain antibodies, Fab, F(ab′), (Fab)₂,scFv, Fd, Fv, or a full-length antibody, e.g. of an IgG type (e.g., anIgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgMantibody. The term “full length antibody” can be used to refer to anyantibody molecule comprising at least most of the Fc domain and otherdomains commonly found in a naturally occurring antibody monomer. Thisphrase is used herein to emphasize that a particular antibody moleculeis not an antibody fragment.

The term “antibody” shall specifically include antibodies in theisolated form, e.g. that are substantially free of other antibodiesdirected against different target antigens or comprising a differentstructural arrangement of antibody domains. Still, an isolated antibodymay be comprised in a combination preparation, containing a combinationof the isolated antibody, e.g. with at least one other antibody, such asmonoclonal antibodies or antibody fragments having differentspecificities.

The term “antibody” shall apply to antibodies of animal origin,including human species, such as mammalian, including human, murine,rabbit, goat, lama, cow and horse, or avian, such as hen.

The term “antibody” further applies to chimeric antibodies withsequences of origin of different species, such as sequences of murineand human origin.

The term “chimeric” as used with respect to an antibody refers to thoseantibodies wherein one portion of each of the amino acid sequences ofheavy and light chains is homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular class, while the remaining segment of the chain is homologousto corresponding sequences in another species or class. Typically thevariable region of both light and heavy chains mimics the variableregions of antibodies derived from one species of mammals, while theconstant portions are homologous to sequences of antibodies derived fromanother. For example, the variable region can be derived from presentlyknown sources using readily available B-cells or hybridomas fromnon-human host organisms in combination with constant regions derivedfrom, for example, human cell preparations.

The term “antibody” further applies to humanized antibodies.

The term “humanized” as used with respect to an antibody refers to amolecule having an antigen binding site that is substantially derivedfrom an immunoglobulin from a non-human species, wherein the remainingimmunoglobulin structure of the molecule is based upon the structureand/or sequence of a human immunoglobulin. The antigen binding site mayeither comprise complete variable domains fused onto constant domains oronly the complementarity determining regions (CDR) grafted ontoappropriate framework regions in the variable domains. Antigen-bindingsites may be wild-type or modified, e.g., by one or more amino acidsubstitutions, preferably modified to resemble human immunoglobulinsmore closely. Some forms of humanized antibodies preserve all CDRsequences (for example a humanized mouse antibody which contains all sixCDRs from the mouse antibody). Other forms have one or more CDRs whichare altered with respect to the original antibody.

The term “antibody” further applies to human antibodies

The term “human” as used with respect to an antibody, is understood toinclude antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibody of theinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs. Human antibodies include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulin.

The term specifically applies to antibodies of any class or subclass.Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to the major classes ofantibodies IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA, and IgA2.

The term further applies to monoclonal or polyclonal antibodies,specifically a recombinant antibody, which term includes all antibodiesand antibody structures that are prepared, expressed, created orisolated by recombinant means, such as antibodies originating fromanimals, e.g., mammalians including human, that comprises genes orsequences from different origin, e.g., chimeric, humanized antibodies,or hybridoma derived antibodies. Further examples refer to antibodiesisolated from a host cell transformed to express the antibody, orantibodies isolated from a recombinant, combinatorial library ofantibodies or antibody domains, or antibodies prepared, expressed,created or isolated by any other means that involve splicing of antibodygene sequences to other DNA sequences.

It is understood that the term “antibody” also refers to derivatives ofan antibody, in particular functionally active derivatives. An antibodyderivative is understood as any combination of one or more antibodydomains or antibodies and/ or a fusion protein, in which any domain ofthe antibody may be fused at any position of one or more other proteins,such as other antibodies, e.g. a binding structure comprising CDR loops,a receptor polypeptide, but also ligands, scaffold proteins, enzymes,toxins and the like. A derivative of the antibody may be obtained byassociation or binding to other substances by various chemicaltechniques such as covalent coupling, electrostatic interaction,di-sulphide bonding etc. The other substances bound to the antibody maybe lipids, carbohydrates, nucleic acids, organic and inorganic moleculesor any combination thereof (e.g. PEG, prodrugs or drugs). In a specificembodiment, the antibody is a derivative comprising an additional tagallowing specific interaction with a biologically acceptable compound.There is not a specific limitation with respect to the tag usable in thepresent invention, as far as it has no or tolerable negative impact onthe binding of the antibody to its target. Examples of suitable tagsinclude His-tag, Myc-tag, FLAG-tag, Strep-tag, Calmodulin-tag, GST-tag,MBP-tag, and S-tag. In another specific embodiment, the antibody is aderivative comprising a label. The term “label” as used herein refers toa detectable compound or composition which is conjugated directly orindirectly to the antibody so as to generate a “labeled” antibody. Thelabel may be detectable by itself, e.g., radioisotope labels orfluorescent labels, or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

The preferred derivatives as described herein are functionally activewith regard to the antigen binding, preferably which have a potency tocombat MDR E. coli and its endotoxin, e.g., as determined in an SBA, OPKor LAL assay, or to protect against bacterial challenge or to neutralizelethal endotoxemia.

Antibodies derived from a parent antibody or antibody sequence areherein particularly understood as mutants or variants obtained by e.g.,in silico or recombinant engineering or else by chemical derivatizationor synthesis.

Specifically, an antibody derived from an antibody of the invention maycomprise at least one or more of the CDR regions or CDR variants thereofbeing functionally active in differentially binding to the O25b antigen,e.g., specifically or selectively binding the O25b antigen.

It is understood that the term “antibody” also refers to variants of anantibody.

The term “variant” shall particularly refer to antibodies, such asmutant antibodies or fragments of antibodies, e.g. obtained bymutagenesis methods, in particular to delete, exchange, introduceinserts into a specific antibody amino acid sequence or region orchemically derivatize an amino acid sequence, e.g. in the constantdomains to engineer the antibody stability, effector function orhalf-life, or in the variable domains to improve antigen-bindingproperties, e.g. by affinity maturation techniques. Any of the knownmutagenesis methods may be employed, including point mutations atdesired positions, e.g., obtained by randomization techniques. In somecases positions are chosen randomly, e.g. with either any of thepossible amino acids or a selection of preferred amino acids torandomize the antibody sequences. The term “mutagenesis” refers to anyart recognized technique for altering a polynucleotide or polypeptidesequence. Preferred types of mutagenesis include error prone PCRmutagenesis, saturation mutagenesis, or other site directed mutagenesis.

The term “variant” shall specifically encompass functionally activevariants.

The term “functionally active variant” of an antibody as used herein,means a sequence resulting from modification of this sequence (a parentantibody or a parent sequence), e.g. by insertion, deletion orsubstitution of one or more amino acids, such as by recombinationtechniques or chemical derivatization of one or more amino acid residuesin the amino acid sequence, or nucleotides within the nucleotidesequence, or at either or both of the distal ends of the sequence, andwhich modification does not affect (in particular impair) the activityof this sequence. In the case of a binding site having specificity to aselected target antigen, the functionally active variant of an antibodywould still have the predetermined binding specificity, though thiscould be changed, e.g. to change the fine specificity to a specificepitope, the affinity, the avidity, the Kon or Koff rate, etc.Specifically, the functionally active variants of an antibody of theinvention have the potency to bind O25b antigen and the specificity orselectivity to preferentially bind to the O25b antigen relative to otherantigens of E. coli, e.g., binding to O25b and not binding to the O25aantigen of E. coli, or not significantly binding the O25a antigen, orcross-specifically binding both, O25b and O25a antigens, but not bindingto other antigens of E. coli.

Functionally active variants may be obtained, e.g. by changing thesequence of a parent antibody, e.g. an antibody comprising the samebinding site as the antibody designated 6D1-1B2, 8A1-1G8, 8D5-1G10, or8D10-C8, but with modifications within an antibody region besides thebinding site, or derived from a parent antibody, which is any of the6D1-1B2, 8A1-1G8, 8D5-1G10, or 8D10-C8 antibodies, by a modificationwithin the binding site but that does not impair the antigen binding,and preferably would have a biological activity similar to the parentantibody, including the ability to specifically or selectively bind O25bantigen, e.g. binding to O25b and not binding to the O25a antigen of E.coli, or not significantly binding the O25a antigen, orcross-specifically binding both, O25b and O25a antigens, but not bindingto other antigens of E. coli. Optionally, the functionally activevariants may further include a potency of complement mediated killing inan SBA assay, and/ or optionally further include a potency of anantibody mediated phagocytosis in an OPK assay, and/ or optionallyfurther include endotoxin neutralization function in a LAL assay, e.g.,with substantially the same biological activity, as determined by thespecific binding assay or functional test to target MDR E. coli.

For example, the functionally active variants of antibodies 6D1-1B2 and8A1-1G8 have substantially the same and similar binding affinities asthe 8D5-1G10 antibody (see table below).

6D1-1B2 8A1-1G8 8D5-1G10 KD (nM) Kon (1/Ms) kdis (1/s) KD (nM) Kon(1/Ms) Kdis (1/s) KD (nM) Kon (1/Ms) Kdis (1/s) 0.6 8.43E+04 < 5.0 E-054.72 4.01E+04 1.89E-04 1.76 4.23E+04 7.44E-05

The term “substantially the same biological activity” as used hereinrefers to the activity as indicated by substantially the same activitybeing at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98% or even at least 100% or at least 110%,or at least 120%, or at least 130%, or at least 140%, or at least 150%,or at least 160%, or at least 170%, or at least 180%, or at least 190%,e.g., up to 200% of the activity as determined for the parent antibody.

The preferred variants or derivatives as described herein arefunctionally active with regard to the antigen binding, preferably whichhave a potency to specifically bind O25b antigen, and not binding toother antigens of E. coli, e.g. binding to O25b and not binding to theO25a antigen of E. coli, or not significantly binding the O25a antigen,or cross-specifically binding both, O25b and O25 antigens, e.g.preferentially binding the O25b antigen relative to O25a, or binding theO25b with higher affinity as compared to current polyclonal typing seraraised against O25 (025a) strains. Preferred variants are not binding toother antigens of E. coli, with a Kd value difference of at least 2logs, preferably at least 3 logs, and optionally further including apotency of complement mediated killing in an SBA assay, e.g. to achievesignificant reduction in bacterial counts relative to control samplesnot containing the antibody, and/ or optionally further including apotency of an antibody mediated phagocytosis in an OPK assay, such as toachieve significant reduction in bacterial counts relative to controlsamples not containing the antibody, and/ or optionally furtherincluding endotoxin neutralization function in a LAL or TLR4 signallingassay, such as to achieve significant reduction in free LPS relative tocontrol samples not containing the antibody, e.g. with substantially thesame biological activity, as determined by the specific binding assay orfunctional test to target MDR E. coli. The significant reduction ofanalytes in the various assays typically means the reduction of at least50%, preferably at least 60%, 70%, 80%, 90%, 95% or 98% up to completereduction of about 100% (+/- 1%).

In a preferred embodiment the functionally active variant of a parentantibody

-   a) is a biologically active fragment of the antibody, the fragment    comprising at least 50% of the sequence of the molecule, preferably    at least 60%, at least 70%, at least 80%, at least 90%, or at least    95% and most preferably at least 97%, 98% or 99%;-   b) is derived from the antibody by at least one amino acid    substitution, addition and/or deletion, wherein the functionally    active variant has a sequence identity to the molecule or part of    it, such as an antibody of at least 50% sequence identity,    preferably at least 60%, more preferably at least 70%, more    preferably at least 80%, still more preferably at least 90%, even    more preferably at least 95% and most preferably at least 97%, 98%    or 99%; and/or-   c) consists of the antibody or a functionally active variant thereof    and additionally at least one amino acid or nucleotide heterologous    to the polypeptide or the nucleotide sequence.

In one preferred embodiment of the invention, the functionally activevariant of the antibody according to the invention is essentiallyidentical to the variant described above, but differs from itspolypeptide or the nucleotide sequence, respectively, in that it isderived from a homologous sequence of a different species. These arereferred to as naturally occurring variants or analogs.

The term “functionally active variant” also includes naturally occurringallelic variants, as well as mutants or any other non-naturallyoccurring variants. As is known in the art, an allelic variant is analternate form of a (poly) peptide that is characterized as having asubstitution, deletion, or addition of one or more amino acids that doesessentially not alter the biological function of the polypeptide.

Functionally active variants may be obtained by sequence alterations inthe polypeptide or the nucleotide sequence, e.g., by one or more-pointmutations, wherein the sequence alterations retains a function of theunaltered polypeptide or the nucleotide sequence, when used incombination of the invention. Such sequence alterations can include, butare not limited to, (conservative) substitutions, additions, deletions,mutations and insertions.

Specific functionally active variants are CDR variants. A CDR variantincludes an amino acid sequence modified by at least one amino acid inthe CDR region, wherein said modification can be a chemical or a partialalteration of the amino acid sequence, which modification permits thevariant to retain the biological characteristics of the unmodifiedsequence. A partial alteration of the CDR amino acid sequence may be bydeletion or substitution of one to several amino acids, e.g. 1, 2, 3, 4or 5 amino acids, or by addition or insertion of one to several aminoacids, e.g. 1, 2, 3, 4 or 5 amino acids, or by a chemical derivatizationof one to several amino acids, e.g. 1, 2, 3, 4 or 5 amino acids, orcombination thereof. The substitutions in amino acid residues may beconservative substitutions, for example, substituting one hydrophobicamino acid for an alternative hydrophobic amino acid.

Conservative substitutions are those that take place within a family ofamino acids that are related in their side chains and chemicalproperties. Examples of such families are amino acids with basic sidechains, with acidic side chains, with non-polar aliphatic side chains,with non-polar aromatic side chains, with uncharged polar side chains,with small side chains, with large side chains etc.

A point mutation is particularly understood as the engineering of apolynucleotide that results in the expression of an amino acid sequencethat differs from the non-engineered amino acid sequence in thesubstitution or exchange, deletion or insertion of one or more single(nonconsecutive) or doublets of amino acids for different amino acids.

Preferred point mutations refer to the exchange of amino acids of thesame polarity and/or charge. In this regard, amino acids refer to twentynaturally occurring amino acids encoded by sixty-four triplet codons.These 20 amino acids can be split into those that have neutral charges,positive charges, and negative charges:

The “neutral” amino acids are shown below along with their respectivethree-letter and single-letter code and polarity:

-   Alanine: (Ala, A) nonpolar, neutral;-   Asparagine: (Asn, N) polar, neutral;-   Cysteine: (Cys, C) nonpolar, neutral;-   Glutamine: (Gln, Q) polar, neutral;-   Glycine: (Gly, G) nonpolar, neutral;-   Isoleucine: (Ile, I) nonpolar, neutral;-   Leucine: (Leu, L) nonpolar, neutral;-   Methionine: (Met, M) nonpolar, neutral;-   Phenylalanine: (Phe, F) nonpolar, neutral;-   Proline: (Pro, P) nonpolar, neutral;-   Serine: (Ser, S) polar, neutral;-   Threonine: (Thr, T) polar, neutral;-   Tryptophan: (Trp, W) nonpolar, neutral;-   Tyrosine: (Tyr, Y) polar, neutral;-   Valine: (Val, V) nonpolar, neutral; and-   Histidine: (His, H) polar, positive (10%) neutral (90%).

The “positively” charged amino acids are:

-   Arginine: (Arg, R) polar, positive; and-   Lysine: (Lys, K) polar, positive.

The “negatively” charged amino acids are:

-   Aspartic acid: (Asp, D) polar, negative; and-   Glutamic acid: (Glu, E) polar, negative.

“Percent (%) amino acid sequence identity” with respect to the antibodysequences and homologs described herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific polypeptide sequence, after aligningthe sequence and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

An antibody variant is specifically understood to include homologs,analogs, fragments, modifications or variants with a specificglycosylation pattern, e.g., produced by glycoengineering, which arefunctional and may serve as functional equivalents, e.g., binding to thespecific targets and with functional properties.

An antibody of the present invention may or may not exhibit Fc effectorfunction. Preferably the antibody exhibits Fc effector function and isfunctionally active in an SBA and/or OPK assay. Specific antibodies maybe devoid of an active Fc moiety, thus, either composed of antibodydomains that do not contain an Fc part of an antibody or that do notcontain an Fcgamma receptor binding site, or comprising antibody domainslacking Fc effector function, e.g. by modifications to reduce Fceffector functions. Alternative antibodies may be engineered toincorporate modifications to increase Fc effector functions, inparticular to enhance OPK and/or SBA activity.

Such modifications may be affected by mutagenesis, e.g., mutations inthe Fcgamma receptor binding site or by derivatives or agents tointerfere with ADCC and/or CDC activity of an antibody format, so toachieve reduction or increase of Fc effector function.

A significant reduction of Fc effector function is typically understoodto refer to Fc effector function of less than 10% of the unmodified(wild-type) format, preferably less than 5%, as measured by ADCC and/orCDC activity. A significant increase of Fc effector function istypically understood to refer to an increase in Fc effector function ofat least 10% of the unmodified (wild-type) format, preferably at least20%, 30%, 40% or 50%, as measured by ADCC and/or CDC activity.

The term “glycoengineered” variants with respect to antibody sequencesshall refer to glycosylation variants having modified immunogenicproperties, ADCC and/ or CDC as a result of the glycoengineering. Allantibodies contain carbohydrate structures at conserved positions in theheavy chain constant regions, with each isotype possessing a distinctarray of N-linked carbohydrate structures, which variably affect proteinassembly, secretion or functional activity. IgG1 type antibodies areglycoproteins that have a conserved N linked glycosylation site atAsn297 in each CH2 domain. The two complex bi-antennary oligosaccharidesattached to Asn297 are buried between the CH2 domains, forming extensivecontacts with the polypeptide backbone, and their presence is essentialfor the antibody to mediate effector functions such as antibodydependent complement mediated bacterial killing or uptake by phagocyticcells. Removal of N-Glycan at N297, e.g., through mutating N297, e.g.,to A, or T299 typically results in aglycosylated antibody formats withreduced effector function.

Major differences in antibody glycosylation occur between cell lines,and even minor differences are seen for a given cell line grown underdifferent culture conditions. Expression in bacterial cells typicallyprovides for an aglycosylated antibody. CHO cells withtetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GIcNAc, wasreported to have improved functional (ADCC) activity (Umana et al.,1999, Nature Biotech. 17:176-180). In addition to the choice of hostcells, factors that affect glycosylation during recombinant productionof antibodies include growth mode, media formulation, culture density,oxygenation, pH, purification schemes and the like.

The term “antigen-binding site” or “binding site” refers to the part ofan antibody that participates in antigen binding. The antigen bindingsite is formed by amino acid residues of the N-terminal variable (“V”)regions of the heavy (“H”) and/or light (“L”) chains, or the variabledomains thereof. Three highly divergent stretches within the V regionsof the heavy and light chains, referred to as “hypervariable regions”,are interposed between more conserved flanking stretches known asframework regions, The antigen-binding site provides for a surface thatis complementary to the three-dimensional surface of a bound epitope orantigen, and the hypervariable regions are referred to as“complementarity-determining regions”, or “CDRs.” The binding siteincorporated in the CDR is herein also called “CDR binding site”.

The term “antigen” as used herein interchangeably with the terms“target” or “target antigen” shall refer to a whole target molecule or afragment of such molecule recognized by an antibody binding site.Specifically, substructures of an antigen, e.g. a polypeptide orcarbohydrate structure, generally referred to as “epitopes”, e.g. B-cellepitopes or T-cell epitope, which are immunologically relevant, may berecognized by such binding site. Specific antigens like the O25b or O25aantigens are provided as isolated antigens, or else in the form of E.coli cells or cell fractions.

The term “epitope” as used herein shall in particular refer to amolecular structure which may completely make up a specific bindingpartner or be part of a specific binding partner to a binding site of anantibody. An epitope may either be occurring in nature or artificial,and composed of a carbohydrate, a peptidic structure, a fatty acid, anorganic, biochemical or inorganic substance or derivatives thereof andany combinations thereof. If an epitope is comprised in a peptidicstructure, such as a peptide, a polypeptide or a protein, it willusually include at least 3 amino acids, preferably 5 to 40 amino acids,and more preferably between about 10-20 amino acids. Epitopes can beeither linear or conformational epitopes. A linear epitope is comprisedof a single segment of a primary sequence of a polypeptide orcarbohydrate chain. Linear epitopes can be contiguous or overlapping.Conformational epitopes are comprised of amino acids or carbohydratesbrought together by folding the polypeptide to form a tertiary structureand the amino acids are not necessarily adjacent to one another in thelinear sequence. Specifically, and with regard to polypeptide antigens aconformational or discontinuous epitope is characterized by the presenceof two or more discrete amino acid residues, separated in the primarysequence, but assembling to a consistent structure on the surface of themolecule when the polypeptide folds into the native protein/antigen.

Herein the term “epitope” shall particularly refer to the single epitoperecognized by an antibody, or the mixture of epitopes comprising epitopevariants, each recognized by an antibody specifically recognizing thetarget, e.g., the epitope specifically recognized by an antibodyselected from the group consisting of antibodies designated as 6D1-1B2,8A1-1G8, 8D5-1G10 and 8D10-C8. Specifically, the epitope targeted by anantibody selected from the group consisting of 6D1-1B2, 8A1-1G8,8D5-1G10 and 8D10-C8 is a carbohydrate epitope.

The term “expression” is understood in the following way. Nucleic acidmolecules containing a desired coding sequence of an expression productsuch as e.g. an antibody as described herein, and control sequences suchas e.g. a promoter in operable linkage, may be used for expressionpurposes. Hosts transformed or transfected with these sequences arecapable of producing the encoded proteins. In order to effecttransformation, the expression system may be included in a vector;however, the relevant DNA may also be integrated into the hostchromosome. Specifically, the term refers to a host cell and compatiblevector under suitable conditions, e.g., for the expression of a proteincoded for by foreign DNA carried by the vector and introduced to thehost cell.

Coding DNA is a DNA sequence that encodes a particular amino acidsequence for a particular polypeptide or protein such as e.g., anantibody. Promoter DNA is a DNA sequence which initiates, regulates, orotherwise mediates or controls the expression of the coding DNA.Promoter DNA and coding DNA may be from the same gene or from differentgenes, and may be from the same or different organisms. Recombinantcloning vectors will often include one or more replication systems forcloning or expression, one or more markers for selection in the host,e.g., antibiotic resistance, and one or more expression cassettes.

“Vectors” used herein are defined as DNA sequences that are required forthe transcription of cloned recombinant nucleotide sequences, i.e., ofrecombinant genes and the translation of their mRNA in a suitable hostorganism.

An “expression cassette” refers to a DNA coding sequence or segment ofDNA that code for an expression product that can be inserted into avector at defined restriction sites. The cassette restriction sites aredesigned to ensure insertion of the cassette in the proper readingframe. Generally, foreign DNA is inserted at one or more restrictionsites of the vector DNA, and then is carried by the vector into a hostcell along with the transmissible vector DNA. A segment or sequence ofDNA having inserted or added DNA, such as an expression vector, can alsobe called a “DNA construct”.

Expression vectors comprise the expression cassette and additionallyusually comprise an origin for autonomous replication in the host cellsor a genome integration site, one or more selectable markers (e.g. anamino acid synthesis gene or a gene conferring resistance to antibioticssuch as zeocin, kanamycin, G418 or hygromycin), a number of restrictionenzyme cleavage sites, a suitable promoter sequence and a transcriptionterminator, which components are operably linked together. The term“vector” as used herein includes autonomously replicating nucleotidesequences as well as genome integrating nucleotide sequences. A commontype of vector is a “plasmid”, which generally is a self-containedmolecule of double-stranded DNA that can readily accept additional(foreign) DNA and which can readily be introduced into a suitable hostcell. A plasmid vector often contains coding DNA and promoter DNA andhas one or more restriction sites suitable for inserting foreign DNA.Specifically, the term “vector” or “plasmid” refers to a vehicle bywhich a DNA or RNA sequence (e.g., a foreign gene) can be introducedinto a host cell, so as to transform the host and promote expression(e.g., transcription and translation) of the introduced sequence.

The term “host cell” as used herein shall refer to primary subject cellstransformed to produce a particular recombinant protein, such as anantibody as described herein, and any progeny thereof. It should beunderstood that not all progeny are exactly identical to the parentalcell (due to deliberate or inadvertent mutations or differences inenvironment), however, such altered progeny are included in these terms,so long as the progeny retain the same functionality as that of theoriginally transformed cell. The term “host cell line” refers to a cellline of host cells as used for expressing a recombinant gene to producerecombinant polypeptides such as recombinant antibodies. The term “cellline” as used herein refers to an established clone of a particular celltype that has acquired the ability to proliferate over a prolongedperiod of time. Such host cell or host cell line may be maintained incell culture and/or cultivated to produce a recombinant polypeptide.

An “immune response” to a composition, e.g., an immunogenic composition,herein also termed “immunogen” comprising an antigen or epitope, or avaccine as described herein is the development in the host or subject ofa cellular- and/or antibody-mediated immune response to the compositionor vaccine of interest. Usually, such a response consists of the subjectproducing antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

A “protective immune response” is understood as therapeutic immuneresponse and refers to an immune response to an antigen derived from apathogen, which in some way prevents, ameliorates, treats or at leastpartially arrests disease symptoms, side effects or progression.Specifically, the protective immune response is triggered that providesa significantly better outcome of an induced or natural infection ortoxin challenge in comparison to that of the non-immune population.

An immunogen or immunogenic composition usually comprises the antigen orepitope and a carrier, which may specifically comprise an adjuvant. Theterm “adjuvant” refers to a compound that when administered inconjunction with an antigen augments and/or redirects the immuneresponse to the antigen, but when administered alone does not generatean immune response to the antigen. Adjuvants can augment an immuneresponse by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages.Exemplary carriers are liposomes or cationic peptides; exemplaryadjuvants are aluminum phosphate or aluminum hydroxide, MF59 or CpGoligonucleotide.

The term “isolated” or “isolation” as used herein with respect to anucleic acid, an antibody or other compound shall refer to such compoundthat has been sufficiently separated from the environment with which itwould naturally be associated, so as to exist in “substantially pure”form. “Isolated” does not necessarily mean the exclusion of artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with the fundamental activity, andthat may be present, for example, due to incomplete purification. Inparticular, isolated nucleic acid molecules of the present invention arealso meant to include those chemically synthesized.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous in the naturally occurring genome of the organismin which it originated. For example, an “isolated nucleic acid” maycomprise a DNA molecule inserted into a vector, such as a plasmid orvirus vector, or integrated into the genomic DNA of a prokaryotic oreukaryotic cell or host organism. When applied to RNA, the term“isolated nucleic acid” refers primarily to an RNA molecule encoded byan isolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been sufficiently separated from othernucleic acids with which it would be associated in its natural state(i.e., in cells or tissues). An “isolated nucleic acid” (either DNA orRNA) may further represent a molecule produced directly by biological orsynthetic means and separated from other components present during itsproduction.

With reference to polypeptides or proteins, such as antibodies orepitopes of the invention, the term “isolated” shall specifically referto compounds that are free or substantially free of material with whichthey are naturally associated such as other compounds with which theyare found in their natural environment, or the environment in which theyare prepared (e g. cell culture) when such preparation is by recombinantDNA technology practiced in vitro or in vivo. Isolated compounds can beformulated with diluents or adjuvants and still for practical purposesbe isolated - for example, the polypeptides or polynucleotides can bemixed with pharmaceutically acceptable carriers or excipients when usedin diagnosis or therapy. In particular, the isolated antibody of theinvention differs from polyclonal serum preparations raised againstO25(a) strains, because it is provided in the isolated and purifiedform, preferably provided in a preparation comprising the isolatedantibody as the only active substance. This does not preclude, however,that the isolated antibody is provided in a combination productcomprising a limited number of further well-defined (isolated)antibodies. Isolated antibodies may as well be provided on a solid,semi-liquid or liquid carrier, such as beads.

The term “neutralizing” or “neutralization” is used herein in thebroadest sense and refers to any molecule that inhibits a pathogen, suchas MDR E.coli from infecting a subject, or to inhibit the pathogen frompromoting infections by producing potent protein toxins, or to inhibitthe toxins from damaging a target cell in a subject, irrespective of themechanism by which neutralization is achieved. Neutralization can beachieved, e.g., by an antibody that inhibits the binding and/orinteraction of the MDR E.coli endotoxin with its cognate receptor ontarget cells (e.g. binding to the TLR4 receptor). Neutralization canfurther occur by removal of endotoxin molecules from the circulation byFc mediated functions.

The neutralization potency is typically determined in a standard assay,e.g., LAL test, where the inhibition of endotoxin’s biological activityis measured, e.g., by colorimetry.

The term “MDR E. coli” is understood in the following way: Infectionswith multi-drug resistant E. coli that are in a significant portion aredue to the ST131-O25b:H4 clonal lineage, which emerged only in the lastdecade and became a globally spread dominant resistant clone.Multi-resistant E. coli is particularly understood as those strainsdemonstrating resistance to three or more classes of antibiotics, e.g.,the following agents/groups: penicillins, cephalosporins, carbapenems,aminoglycosides, tetracyclines, fuoroquinolones, nitrofurantoin,trimethoprim (and its combinations), fosfomycin, polymixins,chloramphenicol, azthreonam, tigecycline.

The acidic capsular polysaccharide (CPS) is a thick, mucous-like, layerof polysaccharide that surrounds most pathogen E. coli. It is, thus,surprising that the specific epitope recognized by an antibody of theinvention would specifically be accessible on both, the encapsulated andthe non-encapsulated MDR E. coli strain.

Antibodies combating or neutralizing MDR E. coli are interfering withthe pathogens and pathogenic reactions, thus able to limit or preventinfection and/ or to ameliorate a disease condition resulting from suchinfection, or to inhibit MDR E. coli pathogenesis, in particulardissemination and replication into or within sterile bodycompartments/sites of the host. In this regard “protective antibodies”are understood herein as antibodies that are responsible for immunity toan infectious agent observed in active or passive immunity. Inparticular, protective antibodies as described herein are possibly usedfor therapeutic purposes, e.g. for prophylaxis or therapy, to prevent,ameliorate, treat or at least partially arrest disease symptoms, sideeffects or progression of disease induced by a pathogen. Specifically,protective antibodies are able to kill or impede replication of live E.coli cells by e.g. inducing serum bactericidal or opsonophagocyticactivities, or remove whole bacterial cells or the LPS molecules thereoffrom the sterile body sites following therapeutic applications (i.e.given on an established infection). Alternatively, prophylacticallyapplied protective antibodies inhibit establishment of an infection(i.e., spread of E. coli from non-sterile sites to sterile bodycompartments) by one of the abovementioned or other mechanisms.

The term “O25b antigen” is herein understood as the LPS O-antigen withstructure elucidated in example 2 and FIG. 3 (a).The structure issimilar, but distinct from that of the O25(a) antigen. O25b is hereinunderstood as a serotype, which is similar, but distinct from O25a (seeFIGS. 3 (a) ).

The term “O25 antigen” is herein understood as the antigen made of thepentasaccharide repeating unit described by Kenne et al. (Kenne L,Lindberg B, Madden JK, Lindberg AA, Gemski P Jr. Structural studies ofthe Escherichia coli O-antigen 25. Carbohydr Res. 28;122(2):249-56,1983). Before identifying the O25b antigen, the term O25 has stood forO25a as described herein (see FIGS. 3 (b) ).

The term “O25a antigen” is herein understood as a synonym for O25antigen.

The term “recombinant” as used herein shall mean “being prepared by orthe result of genetic engineering”. A recombinant host specificallycomprises an expression vector or cloning vector, or it has beengenetically engineered to contain a recombinant nucleic acid sequence,in particular employing nucleotide sequence foreign to the host. Arecombinant protein is produced by expressing a respective recombinantnucleic acid in a host. The term “recombinant antibody”, as used herein,includes antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinantantibodies comprise antibodies engineered to include rearrangements andmutations which occur, for example, during antibody maturation.

As used herein, the term “specificity” or “specific binding” refers to abinding reaction which is determinative of the cognate ligand ofinterest in a heterogeneous population of molecules. Thus, underdesignated conditions (e.g. immunoassay conditions), an antibodyspecifically binds to its particular target and does not bind in asignificant amount to other molecules present in a sample. The specificbinding means that binding is selective in terms of target identity,high, medium or low binding affinity or avidity, as selected. Selectivebinding is usually achieved if the binding constant or binding dynamicsis at least 10 fold different (understood as at least 1 log difference),preferably the difference is at least 100 fold (understood as at least 2logs difference), and more preferred a least 1000 fold (understood as atleast 3 logs difference). The term “specificity” or “specific binding”is also understood to apply to binders which bind to one or moremolecules, e.g., cross-specific binders.

The antibody of the invention specifically is selective in only bindingthe O25b antigen, or preferentially binding the O25b antigen relative tothe O25a antigen, or binding the O25b with higher affinity as comparedto polyclonal serum raised against O25a strains, which serum binds tothe O25b antigen with a low affinity. Thus, the antibody of theinvention may be understood to differentially bind those antigens, e.g.,at least with equal affinity, or more than equal affinity, such as witha different affinity with a Kd difference of at least 1 log, preferablyat least 2 logs, more preferably at least 3 logs. Such antibodyselectively binding to the O25b antigen relative to the O25a antigen ispreferably used for diagnostic or therapeutic purposes. For somediagnostic purposes an antibody is specifically used which only bindsthe O25b antigen in a detectable manner.

Use of the term “having the same specificity”, “having the same bindingsite” or “binding the same epitope” indicates that equivalent monoclonalantibodies exhibit the same or essentially the same, i.e., similarimmunoreaction (binding) characteristics and compete for binding to apre-selected target binding sequence. The relative specificity of anantibody molecule for a particular target can be relatively determinedby competition assays, e.g., as described in Harlow, et al., ANTIBODIES:A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988).

The term “subject” as used herein shall refer to a warm-bloodedmammalian, particularly a human being. In particular the medical use ofthe invention or the respective method of treatment applies to a subjectin need of prophylaxis or treatment of a disease condition associatedwith a MDR E. coli infection or suffering from disease, including earlystage or late stage disease. The term “patient” includes human and othermammalian subjects that receive either prophylactic or therapeutictreatment. The term “treatment” is thus meant to include bothprophylactic and therapeutic treatment.

A subject is e.g., treated for prophylaxis or therapy of MDR E. colidisease conditions. In particular, the subject is treated, which iseither at risk of infection or developing such disease or diseaserecurrence, or a subject that is suffering from such infection and/ ordisease associated with such infection.

Specifically, the term “prophylaxis” refers to preventive measures whichis intended to encompass prevention of the onset of pathogenesis orprophylactic measures to reduce the risk of pathogenesis.

Specifically, the method for treating, preventing, or delaying a diseasecondition in a subject as described herein, is by interfering with thepathogenesis of MDR E. coli as causal agent of the condition.

The term “substantially pure” or “purified” as used herein shall referto a preparation comprising at least 50% (w/w), preferably at least 60%,70%, 80%, 90% or 95% of a compound, such as a nucleic acid molecule oran antibody. Purity is measured by methods appropriate for the compound(e.g., chromatographic methods, polyacrylamide gel electrophoresis, HPLCanalysis, and the like).

The term “therapeutically effective amount”, used herein interchangeablywith any of the terms “effective amount” or “sufficient amount” of acompound, e.g., an antibody or immunogen of the present invention, is aquantity or activity sufficient to, when administered to the subjecteffect beneficial or desired results, including clinical results, and,as such, an effective amount or synonym thereof depends upon the contextin which it is being applied.

An effective amount is intended to mean that amount of a compound thatis sufficient to treat, prevent or inhibit such diseases or disorder. Inthe context of disease, therapeutically effective amounts of theantibody as described herein are specifically used to treat, modulate,attenuate, reverse, or affect a disease or condition that benefits froman inhibition of MDR E. coli pathogenesis, for example, adhesion andcolonization of mucosal surfaces, uncontrolled replication withinsterile body sites, and toxicity of host cells by bacterial products.

The amount of the compound that will correspond to such an effectiveamount will vary depending on various factors, such as the given drug orcompound, the pharmaceutical formulation, the route of administration,the type of disease or disorder, the identity of the subject or hostbeing treated, and the like, but can nevertheless be routinelydetermined by one skilled in the art.

The antibody or the immunogen of the present invention may be usedprophylactically to inhibit onset of MDR E. coli infection, ortherapeutically to treat MDR E. coli infection, particularly MDR E. coliinfections that are known to be refractory or in the case of thespecific subject, have proven refractory to treatment with otherconventional antibiotic therapy.

A therapeutically effective amount of the antibody as described herein,such as provided to a human patient in need thereof, may specifically bein the range of 0.5-500 mg, preferably 1-400 mg, even more preferred upto 300 mg, up to 200 mg, up to 100 mg or up to 10 mg, though higherdoses may be indicated e.g., for treating acute disease conditions.

Moreover, a treatment or prevention regime of a subject with atherapeutically effective amount of the antibody of the presentinvention may consist of a single administration, or alternativelycomprise a series of applications. For example, the antibody may beadministered at least once a year, at least once a half-year or at leastonce a month. However, in another embodiment, the antibody may beadministered to the subject from about one time per week to about adaily administration for a given treatment. The length of the treatmentperiod depends on a variety of factors, such as the severity of thedisease, either acute or chronic disease, the age of the patient, theconcentration and the activity of the antibody format. It will also beappreciated that the effective dosage used for the treatment orprophylaxis may increase or decrease over the course of a particulartreatment or prophylaxis regime. Changes in dosage may result and becomeapparent by standard diagnostic assays known in the art. In someinstances, chronic administration may be required.

An effective amount of an immunogen as described herein, such asprovided to a patient at risk of developing a disease conditionassociated with an MDR E. coli infection, may specifically be in therange of 1-15 mg/kg per dose.

For example, the immunogen may be administered as a first dose followedby one or more booster dose(s), within a certain timeframe, according toa prime-boost immunization scheme to induce a long-lasting, efficaciousimmune response to an MDR E. coli infection. A preferred vaccinationschedule would encompass administration of three doses, e.g. a firstdose on day 0, a second dose on day 5-40, and a third dose on day10-100, preferably on days 0, 28 and 90. According to a preferredaccelerated schedule the administration may be on days 0, 7 and 14.Accelerated schedules may be indicated for prophylaxis, e.g. forpatients facing elective surgery. Usually, alum is used as an adjuvant,e.g., as phosphate or hydroxide.

Therefore, the present subject matter is based on the discovery ofmurine mAbs highly specific to O25b. These antibodies have greatpotential as diagnostic reagents for the identification of MDR strainsbelonging to the ST131 lineage. Furthermore, in particular followinghumanization, these mAbs are suitable to be used for the prophylaxis(e.g., for high-risk groups) and treatment of E. coli infections causedby ST131-O25b:H4 strains.

The O25b and O25 (O25a) carbohydrate antigens were thought to beidentical or very similar based on the fact that immune serum againstO25 is routinely used in the diagnostic identification of E. colistrains expressing O25b antigens. The genetic background of O-antigensynthesis in ST131 strains is not fully elucidated, however, a specificgene within the rƒb cluster (encoding O-antigen synthesis) forms thebasis of PCR based identification of O25b strains. Furthermore, nostructural data supported any difference between O25(a) and O25bantigens so far.

It was, thus, surprising that an antibody of invention couldspecifically bind the O25b antigen, and specifically differentiatebetween O25b and O25a antigens.

In order to confirm the genetic difference between O25b and O25a antigenexpressing E. coli strains, the rƒb cluster encoding O-antigen synthesiswas sequenced from a commercially available strain 81009 (Szijarto etal, FEMS Microbiol Lett, 2012, 332:131-6) using a primer walk methodstarting with oligonucleotides specific to conserved flanking genes: gndand galF. The resulting contig of the rƒb operon is 11,300 bp long andonly partially homologous to that encoding the O25 antigen synthesisenzymes (NCBI accession number GU014554). It turned out that a 2043 bplong segment at the 3′ end of the O25b rƒb operon is non-homologous tothe corresponding region of the O25 rfb operon, where this segment isreplaced by a 6267 bp long sequence encoding fucose synthesis amdtransport.

The structure of the O-specific PS biological repeating unit (RU)present in LPS isolated from E. coli ST131 was analysed in details in apurified fraction built up by the core OS substituting with onerepeating unit (RU). The RU of the LPS ST131 is an O-acetylatedpentasaccharide with the structure depicted on FIG. 3 .

In fact, the RU structure of the ST131 O-PS differs from the LPS O25 RUreported by Kenne et al. (Kenne, Lindberg et al., Carbohydr Res. 1983Oct 28; 122(2):249-56) and to the best of our knowledge it is a newO-serotype among E. coli lipopolysaccharides (Stenutz et al. FEMSMicrobiol Rev. 2006 May; 30(3):382-403. Review). Additionally,preliminary results of MALDI-TOF mass spectrometry and compositionanalyses (sugar and methylation analyses) of a core oligosaccharideisolated from LPS ST131 supported K-12 type, what was previouslyreported by Szijártó V. et al. on the basis of genetic analyses(Szijarto et al, FEMS Microbiol Lett, 2012, 332:131-6). It was shownthat LPS ST131 is consisted of two main core oligosaccharides (OS)glycoforms. The type of glycoform is dependent on the presence orabsence of the O-specific polysaccharide (PS). Prevailing glycoform ofthe unsubstituted core OS is truncated version of K-12 coreoligosaccharide, which is devoid of →7)-α-Hepp-(1→6)-α-Glcpdisaccharide. Presence of that disaccharide is the difference betweenO-PS substituted core OS and nonsubstituted core OS.

According to a specific aspect, there is provided an antibodyselectively binding the O25b specific epitope, e.g. binding the sameepitope as the 8D5-1G10 antibody or any of the antibodies designated as6D1-1B2 or 8A1-1G8, or the 8D10-C8 antibody, which term includesvariants binding to essentially the same epitope; or comprising the samebinding site as the 8D5-1G10 antibody or any of the antibodiesdesignated as 6D1-1B2 or 8A1-1G8, or the 8D10-C8 antibody, which termincludes variants comprising essentially the same binding site. Theantibodies designated as 6D1-1B2, 8A1-1G8, 8D5-1G10 would particularlycomprise a binding site specifically differentiating between the O25bantigen and the O25a antigen, and binding only the O25b antigen. Theantibody designated as 8D10-C8 would particularly comprise a bindingsite cross-specifically binding the O25b and O25a antigens, andpreferentially binding the O25b antigen as compared to the O25a antigen.

Antibodies are said to “bind to the same epitope” or “comprising thesame binding site” or have “essentially the same binding”characteristics, if the antibodies cross-compete so that only oneantibody can bind to the epitope at a given point of time, i.e., oneantibody prevents the binding or modulating effect of the other.

The term “compete” or “cross-compete”, as used herein with regard to anantibody, means that a first antibody, or an antigen-binding portionthereof, binds to an epitope in a manner sufficiently similar to thebinding of a second antibody, or an antigen-binding portion thereof,such that the result of binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope, whether to the same, greater, or lesser extent, the antibodiesare said to “cross-compete” with each other for binding of theirrespective epitope(s). Both competing and cross-competing antibodies areencompassed by the present invention.

Competition herein means a greater relative inhibition than about 30% asdetermined by competition ELISA analysis, e.g. as described in theExamples section. It may be desirable to set a higher threshold ofrelative inhibition as criteria of what is a suitable level ofcompetition in a particular context, e.g., where the competitionanalysis is used to select or screen for new antibodies designed withthe intended function of the binding of O25b. Thus, for example, it ispossible to set criteria for the competitive binding, wherein at least40% relative inhibition is detected, or at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or even at least 100%, before anantibody is considered sufficiently competitive.

Specifically, there is provided an antibody comprising the variableregion of any of the antibodies designated as 6D1-1B2, 8A1-1G8,8D5-1G10, or 8D10-C8, in particular at least one of the CDR sequences,preferably at least two, at least 3, at least 4, at least 5 or at leastsix of the CDR sequences of an antibody selected from the groupconsisting of 6D1-1B2, 8A1-1G8, 8D5-1G10 and 8D10-C8 antibodies, or CDRvariants thereof which are functionally active. More specifically, thereis provided any of the antibodies designated as 6D1-1B2, 8A1-1G8,8D5-1G10, or 8D10-C8.

Specifically, the antibody designated as 8D5-1G10 antibody or 8D10-C8antibody, or any functionally active variant thereof may be producedemploying the deposited material or the respective nucleotide sequencecontained therein, such as one of the plasmids and/or one of thedeposited host cells.

According to a specific aspect, the 8D5-1G10 antibody or a functionallyequivalent variant thereof may be derived from an antibody comprising avariable region encoded by any of the plasmids incorporated in thedeposited host cells under DSM 26763 and/or DSM 26762; e.g., employing apartial or (point) mutated CDR sequence of the deposited material toengineer the specific antibody or any functionally active variantthereof.

According to a further specific aspect, the 8D5-1G10 antibody or afunctionally equivalent variant thereof may be derived from or employingthe variable region of an antibody produced by a host cell depositedunder DSM 26763 and/or DSM 26762; e.g., employing a partial sequence,e.g., one or more of the CDR sequences, of the deposited material toengineer the specific antibody or any functionally active variantthereof.

Specifically, the 6D1-1B2 or 8A1-1G8 antibody variant is a CDR variantof the 8D5-1G10 antibody that is functionally active, e.g., with partialalterations in at least one of the CDR sequences.

According to a specific aspect, the 8D10-C8 antibody or a functionallyequivalent variant thereof may be derived from an antibody comprising avariable region encoded by any of the plasmids incorporated in thedeposited host cells under DSM 28171 and/or DSM 28172; e.g., employing apartial or (point) mutated CDR sequence of the deposited material toengineer the specific antibody or any functionally active variantthereof.

According to a further specific aspect, the 8D10-C8 antibody or afunctionally equivalent variant thereof may be derived from or employingthe variable region of an antibody produced by a host cell depositedunder DSM 28171 and/or DSM 28172; e.g., employing a partial sequence,e.g., one or more of the CDR sequences, of the deposited material toengineer the specific antibody or any functionally active variantthereof.

In certain aspects, the invention provides for such variant antibodies,preferably monoclonal antibodies, most preferably murine, humanized orhuman antibodies, comprising a heavy chain and a light chain, whereinany of the heavy chain or VH variable region or the respective CDRscomprises an amino acid sequence as derived from the respectivedeposited plasmid and/or from the respective deposited host cell.

In certain aspects, the invention provides for such variant antibodies,preferably monoclonal antibodies, most preferably murine, humanized orhuman antibodies, comprising a heavy chain and a light chain, whereinany of the light chain or VL variable region or the respective CDRscomprises an amino acid sequence as derived from the respectivedeposited plasmid and/or from the respective deposited host cell.

In certain aspects, the invention provides for such variant antibodies,preferably monoclonal antibodies, most preferably murine, humanized orhuman antibodies, comprising a heavy chain and a light chain, whereinany of the heavy and light chain, or the VH/VL variable regions, or therespective CDRs comprises an amino acid sequence as derived from therespective deposited plasmids and/or from the respective deposited hostcells.

In certain aspects, the invention also provides for such variantantibodies, comprising the respective binding sequences, such as thevariable sequences and/or the CDR sequences, as derived from thedeposited material, wherein the binding sequences, e.g. any CDRsequence, comprises a sequence that has at least 60%, at least 70%, orat least 80%, or at least 90%, or at least 95%, or at least 99% identityto the amino acid sequence as derived from the deposited material, andwherein the variant is a functionally active variant.

As described herein, in one aspect the invention provides antibodymolecules characterized by, e.g. the ability to compete with monoclonalantibody 8D5-1G10 or 8D10-C8 for binding to the O25b antigen. Any of the6D1-1B2, 8A1-1G8, 8D5-1G10, antibodies is a murine IgG3 antibody and8D10-C8 antibody is a murine IgG2b antibody which carries kappa lightchains, which the inventors isolated and characterized. The variabledomains of the 8D5-1G10 antibody or the 8D10-C8 antibody are expressedby the deposited material as referenced herein. Thus, the bindingcharacteristics as determined by the light chain and heavy chain, andthe VL/VH domains of the 8D5-1G10 antibody or the 8D10-C8 antibody arefully disclosed herewith, enabling its use as a parent antibody or thecomparison with functionally active variants or competing antibodies ofthe invention.

The mature variable domain of the heavy chain of 8D5-1G10 (8D5-1G10-HC)is e.g., produced employing the host cell of DSM 26762, or therespective sequence information of the encoding plasmid incorporatedtherein.

The mature variable domain of the light chain of 8D5-1G10 (8D5-1G10-LC)is e.g., produced employing the host cell of DSM 26763, or therespective sequence information of the encoding plasmid incorporatedtherein.

The mature variable domain of the heavy chain of 8D10-C8 (8D10-C8-HC) ise.g., produced employing the host cell of DSM 28172, or the respectivesequence information of the encoding plasmid incorporated therein.

The mature variable domain of the light chain of 8D10-C8 (8D10-C8-LC) ise.g., produced employing the host cell of DSM 28171, or the respectivesequence information of the encoding plasmid incorporated therein.

The differential binding affinity to preferably bind the O25b antigenrelative to other E. coli antigens, e.g., any carbohydrate antigensother than O25 antigen or any core antigens, is preferably at least10-fold higher, i.e., with a Kd difference of at least 10, preferably atleast 100-fold higher, more preferred at least 1000-fold higher.

The differential binding affinity to preferentially bind the O25bantigen is specifically at least 5-fold, or at least 6-fold, or at least7-fold, or at least 8-fold, or at least 9-fold, or at least 10-foldhigher, in comparison to commercial typing serum, such as the high titerE. coli O25 antiserum from Statens Serum Institut (#81369).

The differential binding affinity to preferentially bind the O25bantigen relative to the O25a antigen is specifically at least equal ormore than equal, e.g. at least 1.5 fold, or at least 2-fold, or at least3-fold, or at least 4-fold, or at least 5 fold, or at least 6-fold, orat least 7-fold, or at least 8-fold, or at least 9-fold, or at least10-fold higher.

Preferred antibodies of the invention are binding any of said individualantigens, in particular the O25b antigen, with a high affinity, inparticular with a high on and/or a low off rate, or a high avidity ofbinding. The binding affinity of an antibody is usually characterized interms of the concentration of the antibody, at which half of the antigenbinding sites are occupied, known as the dissociation constant (Kd, orK_(D)). Usually, a binder is considered a high affinity binder with aKd<10⁻⁷ M, in some cases, e.g., for therapeutic purposes with higheraffinities, e.g., with a Kd<10⁻⁸ M, preferably a Kd<10⁻⁹ M, even morepreferred is a Kd<10⁻¹⁰ M.

Yet, in a particularly preferred embodiment the individual antigenbinding affinities are of medium affinity, e.g., with a Kd of less than10⁻⁶ and up to 10⁻⁷ M, e.g., when binding to at least two antigens.

Medium affinity binders may be provided according to the invention,preferably in conjunction with an affinity maturation process, ifnecessary.

Affinity maturation is the process by which antibodies with increasedaffinity for a target antigen are produced. With structural changes ofan antibody, including amino acid mutagenesis or as a consequence ofsomatic mutation in immunoglobulin gene segments, variants of a bindingsite to an antigen are produced and selected for greater affinities.Affinity matured antibodies may exhibit a several logfold greateraffinity than a parent antibody. Single parent antibodies may be subjectto affinity maturation. Alternatively, pools of antibodies with similarbinding affinity to the target antigen may be considered as parentstructures that are varied to obtain affinity matured single antibodiesor affinity matured pools of such antibodies.

The preferred affinity maturated variant of an antibody according to theinvention exhibits at least a 10-fold increase in affinity of binding,preferably at least a 100-fold increase. The affinity maturation may beemployed in the course of the selection campaigns employing respectivelibraries of parent molecules, either with antibodies having mediumbinding affinity to obtain the antibody of the invention having thespecific target binding property of a binding affinity Kd<10⁻⁷ M.Alternatively, the affinity may be even more increased by affinitymaturation of the antibody according to the invention to obtain the highvalues corresponding to a Kd of less than 10⁻⁸ M or less than 10⁻⁹ M,preferably less than 10⁻¹⁰ M or even less than 10⁻¹¹ M, most preferredin the picomolar range.

A specific aspect refers to an antibody of the invention characterizedby a specific anti-bacterial functional activity, such as complementmediated bacterial killing and opsonophagocytic uptake and killing.

Phagocytic effector cells may be activated through another routeemploying activation of complement. Antibodies that bind to surfaceantigens on microorganisms attract the first component of the complementcascade with their Fc region and initiate activation of the “classical”complement system. This results in the stimulation of phagocyticeffector cells, which ultimately kill the target bacteria by complementand antibody dependent mechanisms (CDC).

According to a specific embodiment, the antibody of the invention has acytotoxic activity in the presence of immune-effector cells as measuredin a standard SBA or OPK assay. A cytotoxic activity as determined byeither of an SBA or OPK assay may be shown for an antibody of theinvention, if there is a significant increase in the percentage ofbacterial killing as compared to a control. The bactericidal activityrelated to SBA or OPK is preferably measured as the absolute percentageincrease, which is preferably higher than 5%, more preferably higherthan 10%, even more preferred higher than 20%, 30%, 40% or 50%.

Antibodies of the present invention may be identified or obtainedemploying a hybridoma method. In such method, a mouse or otherappropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

For example, the antibodies of the present invention may be obtainedfrom source (parent) antibodies, e.g. obtained by immunizing mice with anon-encapsulated mutant of a representative ST131-O25b:H4 strain 81009(e.g. 81009Δkps::kan) by replacing the kps cluster (encoding capsularsynthesis) with a cassette encoding kanamycin resistance. Serum samplesobtained from the mice may then be analyzed, and the spleen of the mouseshowing the highest IgG titer against O25b antigen (in ELISA and WesternBlot) may be used for hybridoma generation. Following sub-cloning,hybridoma clones may be selected, which secreted antibodies specific toO25b antigens as well as bound to the surface of live wild-type E. colistrains expressing O25b antigens. These mAbs may then be purified fromhybridoma supernatants for further testing for its specific binding ofO25b antigen and possibly for its differential binding affinity topreferentially bind O25b antigen relative to O25a antigen, andengineering of antibodies, e.g., for different diagnostic or therapeuticpurposes.

Differentially binding antibodies, herein also called selectiveantibodies, in some instances, emerge through screening against singleantigens. To increase the likelihood of isolating differentially bindingclones one would apply multiple selective pressures by processivelyscreening against the different antigens. Special mAb selectionstrategies employ the O25b and O25a components or other E. coli antigensin an alternating fashion.

The recombinant antigen(s) may be used for selecting antibodies from anantibody library, e.g., a yeast-displayed antibody library.

In either event, selective binding can be further improved by antibodyoptimization methods known in the art. For example, certain regions ofthe variable regions of the immunoglobulin chains described herein maybe subjected to one or more optimization strategies, including lightchain shuffling, destinational mutagenesis, CDR amalgamation, anddirected mutagenesis of selected CDR and/or framework regions.

Screening methods for identifying antibodies with the desired selectivebinding properties may be done by display technologies (using phage,bacterial, yeast or mammalian cells). Reactivity can be assessed basedon ELISA, Western blotting or surface staining with flow cytometry,e.g., using standard assays.

Once differentially binding antibodies with the desired properties havebeen identified, the dominant epitope or epitopes recognized by theantibodies may be determined. Methods for epitope mapping are well-knownin the art and are disclosed, for example, in Epitope Mapping: APractical Approach, Westwood and Hay, eds., Oxford University Press,2001.

Epitope mapping concerns the identification of the epitope to which anantibody binds. There are many methods known to those of skill in theart for determining the location of epitopes on proteins, includingcrystallography analysis of the antibody-antigen complex, competitionassays, gene fragment expression assays, and synthetic peptide-basedassays. An antibody that “binds the same epitope” as a referenceantibody is herein understood in the following way. When two antibodiesrecognize epitopes that are identical or sterically overlappingepitopes, the antibodies are referred to as binding the same oressentially the same or substantially the same epitopes. A commonly usedmethod for determining whether two antibodies bind to identical orsterically overlapping epitopes is the competition assay, which can beconfigured in all number of different formats, using either labeledantigen or labeled antibody. Usually, an antigen is immobilized on a96-well plate, and the ability of unlabeled antibodies to block thebinding of labeled antibodies is measured using radioactive or enzymelabels.

Once antibodies with the desired differentially binding properties areidentified, such antibodies, including antibody fragments can beproduced by methods well-known in the art, including, for example,hybridoma techniques or recombinant DNA technology.

Recombinant monoclonal antibodies can, for example, be produced byisolating the DNA encoding the required antibody chains and transfectinga recombinant host cell with the coding sequences for expression, usingwell known recombinant expression vectors, e.g., the plasmids of theinvention or expression cassette(s) comprising the nucleotide sequencesencoding the antibody sequences. Recombinant host cells can beprokaryotic and eukaryotic cells, such as those described above.

According to a specific aspect, the nucleotide sequence may be used forgenetic manipulation to humanize the antibody or to improve theaffinity, or other characteristics of the antibody. For example, theconstant region may be engineered to more nearly resemble human constantregions to avoid immune response, if the antibody is used in clinicaltrials and treatments in humans. It may be desirable to geneticallymanipulate the antibody sequence to obtain greater affinity to thetarget O25b and greater efficacy against MDR E. coli. It will beapparent to one of skill in the art that one or more polynucleotidechanges can be made to the antibody and still maintain its bindingability to the target O25b.

The production of antibody molecules, by various means, is generallywell understood. U.S. Pat. 6331415 (Cabilly et al.), for example,describes a method for the recombinant production of antibodies wherethe heavy and light chains are expressed simultaneously from a singlevector or from two separate vectors in a single cell. Wibbenmeyer etal., (1999, Biochim Biophys Acta 1430(2):191 -202) and Lee and Kwak(2003, J. Biotechnology 101 :189-198) describe the production ofmonoclonal antibodies from separately produced heavy and light chains,using plasmids expressed in separate cultures of E. coli. Various othertechniques relevant to the production of antibodies are provided in,e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

If desired, the antibody of the invention, e.g., the 8D5-1G10 antibodyor the 8D10-C8 antibody, or the respective binding site or CDR may besequenced and the polynucleotide sequence or a sequence variant ormutant thereof may then be cloned into a vector for expression orpropagation. The sequence encoding the antibody may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. Production of recombinant monoclonal antibodies in cellculture can be carried out through cloning of antibody genes from Bcells by means known in the art.

In another aspect, the invention provides an isolated nucleic acidcomprising a sequence that codes for production of the recombinantantibody of the present invention.

In another aspect, the invention provides an isolated nucleic acidcomprising a sequence that codes for production of the recombinantepitope of the present invention, or a molecule comprising such epitopeof the present invention. However, the epitope of the invention may alsobe synthetically produced, e.g., through any of the synthesis methodswell-known in the art.

An antibody or epitope encoding nucleic acid can have any suitablecharacteristics and comprise any suitable features or combinationsthereof. Thus, for example, an antibody or epitope encoding nucleic acidmay be in the form of DNA, RNA, or a hybrid thereof, and may includenon-naturally-occurring bases, a modified backbone, e.g., aphosphothioate backbone that promotes stability of the nucleic acid, orboth. The nucleic acid advantageously may be incorporated in anexpression cassette, vector or plasmid of the invention, comprisingfeatures that promote desired expression, replication, and/or selectionin target host cell(s). Examples of such features include an origin ofreplication component, a selection gene component, a promoter component,an enhancer element component, a polyadenylation sequence component, atermination component, and the like, numerous suitable examples of whichare known.

The present disclosure further provides the recombinant DNA constructscomprising one or more of the nucleotide sequences described herein.These recombinant constructs are used in connection with a vector, suchas a plasmid, phagemid, phage or viral vector, into which a DNA moleculeencoding any disclosed antibody is inserted.

Monoclonal antibodies are produced using any method that producesantibody molecules by continuous cell lines in culture. Examples ofsuitable methods for preparing monoclonal antibodies include thehybridoma methods of Kohler et al. (1975, Nature 256:495-497) and thehuman B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; andBrodeur et al., Monoclonal Antibody Production Techniques andApplications pp 51-63 (Marcel Dekker, Inc., 1987)).

The invention moreover provides pharmaceutical compositions whichcomprise an antibody or an immunogen as described herein and apharmaceutically acceptable carrier or excipient. These pharmaceuticalcompositions can be administered in accordance with the presentinvention as a bolus injection or infusion or by continuous infusion.Pharmaceutical carriers suitable for facilitating such means ofadministration are well known in the art.

Pharmaceutically acceptable carriers generally include any and allsuitable solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible with an antibody or relatedcomposition or combination provided by the invention. Further examplesof pharmaceutically acceptable carriers include sterile water, saline,phosphate buffered saline, dextrose, glycerol, ethanol, and the like, aswell as combinations of any thereof.

In one such aspect, an antibody can be combined with one or morecarriers appropriate a desired route of administration, antibodies maybe, e.g. admixed with any of lactose, sucrose, starch, cellulose estersof alkanoic acids, stearic acid, talc, magnesium stearate, magnesiumoxide, sodium and calcium salts of phosphoric and sulphuric acids,acacia, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinylalcohol, and optionally further tableted or encapsulated forconventional administration. Alternatively, an antibody may be dissolvedin saline, water, polyethylene glycol, propylene glycol, carboxymethylcellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseedoil, sesame oil, tragacanth gum, and/or various buffers. Other carriers,adjuvants, and modes of administration are well known in thepharmaceutical arts. A carrier may include a controlled release materialor time delay material, such as glyceryl monostearate or glyceryldistearate alone or with a wax, or other materials well known in theart.

Additional pharmaceutically acceptable carriers are known in the art anddescribed in, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES. Liquidformulations can be solutions, emulsions or suspensions and can includeexcipients such as suspending agents, solubilizers, surfactants,preservatives, and chelating agents.

Pharmaceutical compositions are contemplated wherein an antibody orimmunogen of the present invention and one or more therapeuticallyactive agents are formulated. Stable formulations of the antibody orimmunogen of the present invention are prepared for storage by mixingsaid immunoglobulin having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers, in theform of lyophilized formulations or aqueous solutions. The formulationsto be used for in vivo administration are specifically sterile,preferably in the form of a sterile aqueous solution. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods. The antibody and other therapeutically active agents disclosedherein may also be formulated as immunoliposomes, and/or entrapped inmicrocapsules.

Administration of the pharmaceutical composition comprising an antibodyor immunogen of the present invention, may be done in a variety of ways,including orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, mucosal, topically, e.g., gels, salves,lotions, creams, etc., intraperitoneally, intramuscularly,intrapulmonary, e.g., employing inhalable technology or pulmonarydelivery systems, vaginally, parenterally, rectally, or intraocularly.

Exemplary formulations as used for parenteral administration includethose suitable for subcutaneous, intramuscular or intravenous injectionas, for example, a sterile solution, emulsion or suspension.

In one embodiment, the antibody or immunogen of the present invention isthe only therapeutically active agent administered to a subject, e.g.,as a disease modifying or preventing monotherapy.

Alternatively, the antibody or immunogen of the present invention isadministered in combination with one or more other therapeutic orprophylactic agents, including but not limited to standard treatment,e.g., antibiotics, steroid and non-steroid inhibitors of inflammation,and/or other antibody-based therapy, e.g., employing anti-bacterial oranti-inflammatory agents.

A combination therapy is particularly employing a standard regimen,e.g., as used for treating MDR E. coli infection. This may includeantibiotics, e.g., tygecycline, linezolide, methicillin and/orvancomycin.

In a combination therapy, the antibody may be administered as a mixture,or concomitantly with one or more other therapeutic regimens, e.g.,either before, simultaneously or after concomitant therapy.

Prophylactic administration of immunogens in some cases may employ avaccine comprising the immunogen of the present invention, i.e. amonovalent vaccine. Yet, a multivalent vaccine comprising differentimmunogens to induce an immune response against the same or differenttarget pathogens may be used.

The biological properties of the antibody, the immunogen or therespective pharmaceutical preparations of the invention may becharacterized ex vivo in cell, tissue, and whole organism experiments.As is known in the art, drugs are often tested in vivo in animals,including but not limited to mice, rats, rabbits, dogs, cats, pigs, andmonkeys, in order to measure a drug’s efficacy for treatment against adisease or disease model, or to measure a drug’s pharmacokinetics,pharmacodynamics, toxicity, and other properties. The animals may bereferred to as disease models. Therapeutics are often tested in mice,including but not limited to nude mice, SCID mice, xenograft mice, andtransgenic mice (including knockins and knockouts). Such experimentationmay provide meaningful data for determination of the potential of theantibody to be used as a therapeutic or as a prophylactic with theappropriate half-life, effector function, (cross-) neutralizing activityand/or immune response upon active or passive immunotherapy. Anyorganism, preferably mammals, may be used for testing. For examplebecause of their genetic similarity to humans, primates, monkeys can besuitable therapeutic models, and thus may be used to test the efficacy,toxicity, pharmacokinetics, pharmacodynamics, half-life, or otherproperty of the subject agent or composition. Tests in humans areultimately required for approval as drugs, and thus of course theseexperiments are contemplated. Thus, the antibody, immunogen andrespective pharmaceutical compositions of the present invention may betested in humans to determine their therapeutic or prophylacticefficacy, toxicity, immunogenicity, pharmacokinetics, and/or otherclinical properties.

The invention also provides the subject antibody of the invention fordiagnostic purposes, e.g., for use in methods of detecting andquantitatively determining the concentration of a bacterial load orantibody as immunoreagent or target in a biological fluid sample.

The invention also provides methods for detecting the degree of sepsisor MDR E. coli infection in a biological sample, e.g., the load of asample with MDR E. coli, such as a body fluid, comprising the step ofcontacting the sample with an antibody of the invention. The antibody ofthe invention may be employed in any known assay method, such ascompetitive binding assays, direct and indirect sandwich assays,immunoprecipitation assays and enzyme-linked immunosorbent assays(ELISA).

The preferred diagnostic assay is performed as follows. Target antigenspecific antibodies are immobilized on latex beads that are incubatedwith bacteria present in or isolated from body fluids. Positive reactioncan be detected by naked eye due to the aggregation of the colored latexbeads in the presence of the corresponding cognate antigen expressed onthe surface of the bacteria.

A body fluid as used according to the present invention includesbiological samples of a subject, such as tissue extract, urine, blood,serum, stool and phlegm.

In one embodiment the method comprises contacting a solid support withan excess of a certain type of antibody fragment which specificallyforms a complex with the target, under conditions permitting theantibody to attach to the surface of the solid support. The resultingsolid support to which the antibody is attached is then contacted with abiological fluid sample so that the target in the biological fluid bindsto the antibody and forms a target-antibody complex. The complex can belabeled with a detectable marker. Alternatively, either the target orthe antibody can be labeled before the formation the complex. Forexample, a detectable marker (label) can be conjugated to the antibody.The complex then can be detected and quantitatively determined therebydetecting and quantitatively determining the concentration of the targetin the biological fluid sample.

For particular applications the antibody of the invention is conjugatedto a label or reporter molecule, selected from the group consisting oforganic molecules, enzyme labels, radioactive labels, colored labels,fluorescent labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, colloidal gold andmixtures thereof. Antibodies conjugated to labels or reporter moleculesmay be used, for instance, in assay systems or diagnostic methods, e.g.,to diagnose MDR E. coli infection or disease conditions associatedtherewith.

The antibody of the invention may be conjugated to other molecules whichallow the simple detection of said conjugate in, for instance, bindingassays (e.g. ELISA) and binding studies.

Another aspect of the present invention provides a kit comprising anantibody, which may include, in addition to one or more antibodies,various diagnostic or therapeutic agents. A kit may also includeinstructions for use in a diagnostic or therapeutic method. Suchinstructions can be, for example, provided on a device included in thekit, e.g. tools or a device to prepare a biological sample fordiagnostic purposes, such as separating a cell and/or protein containingfraction before determining the MDR E. coli load to diagnose a disease.Advantageously, such a kit includes an antibody and a diagnostic agentor reagent that can be used in one or more of the various diagnosticmethods described herein. In another preferred embodiment, the kitincludes an antibody, e.g. in the lyophilized form, optionally includinginstructions and a medium to reconstitute the lyophilizate, and/or incombination with pharmaceutically acceptable carrier(s) that can bemixed before use to form an injectable composition for near termadministration.

The antibodies designated 8D5-1G10 and 8D10-C8, specifically any of theantibody light chains and/or heavy chains, is further characterized bythe biological material deposited at the DSMZ - Deutsche Sammlung vonMikroorganismen und Zellkulturen, Mascheroder Weg 1b / Inhoffenstraβe7B, 38124 Braunschweig (DE).

The deposits refer to transformed E. coli cultures, each containing aplasmid cloned with an insert of a gene of interest. The genes ofinterest are the variable domains of the heavy and light chains of themouse monoclonal antibody 8D5-1G10 (IgG3), and the heavy and lightchains of the mouse monoclonal antibody 8D10-C8 (IgG2b).

DSM 26763 is an E. coli host cell transformed with a plasmid comprisingthe variable domain coding sequence of the 8D5-1G10 light chain(8D5-1G10-LC). Escherichia coli 8D5-1G10-VL = DSM 26763, depositiondate: January 15th, 2013; depositor: Arsanis Biosciences GmbH, Vienna,Austria.

DSM 26762 is an E. coli host cell transformed with a plasmid comprisingthe variable domain coding sequence of the 8D5-1G10 heavy chain(8D5-1G10-HC). Escherichia coli 8D5-1G10-VH = DSM 26762, depositiondate: January 15th, 2013; depositor: Arsanis Biosciences GmbH, Vienna,Austria.

DSM 28171 is an E. coli host cell transformed with a plasmid comprisingthe variable domain coding sequence of the 8D10-C8 light chain(8D10-C8-LC). Escherichia coli 8D10-C8-VL = DSM 28171, deposition date:December 11th 2013; depositor: Arsanis Biosciences GmbH, Vienna,Austria.

DSM 28172 is an E. coli host cell transformed with a plasmid comprisingthe variable domain coding sequence of the 8D10-C8 heavy chain(8D10-C8-HC). Escherichia coli 8D10-C8-VH = DSM 28172, deposition date:December 11th 2013; depositor: Arsanis Biosciences GmbH, Vienna,Austria.

The subject matter of the following definitions is consideredembodiments of the present invention:

1. An isolated antibody that specifically binds to O25b antigen of multidrug resistant (MDR) E. coli strains.

2. Antibody according to definition 1, which is cross-specific to bindthe O25b and O25 antigens and/or preferentially binds to the O25bantigen relative to an O25a antigen of E. coli, preferably with a higheraffinity as compared to binding the O25b antigen by a polyclonal serumraised against O25 (now known and herein referred to as O25a) E. colistrains as determined by immunoassay, compared to polyclonal typingserum raised against O25a strains, preferably wherein the antibody hasat least equal affinity to both, the O25b and the O25a antigens, asdetermined by immunoassay, preferably immunoblotting, ELISA or otherimmunological methods.

3. Antibody according to definition 1 or 2, wherein the O25b antigen isprevalent in one or more ST131-O25b:H4 strains.

4. Antibody according to any of definitions 1 to 3, wherein the epitoperecognized by the antibody is present on the surface of encapsulated andnon-encapsulated ST131-O25b:H4 strains.

5. Antibody according to any of definitions 1 to 4, which has a bindingsite of a full-length monoclonal antibody or an antibody fragmentthereof comprising at least one antibody domain incorporating a bindingsite, which antibody is preferably an antibody selected from the groupconsisting of murine, lama, rabbit, goat cow, chimeric, humanized orhuman antibodies, heavy-chain antibodies, Fab, Fd, scFv andsingle-domain antibodies like VH, VHH or VL, preferably a human IgG1antibody.

6. Antibody according to any of definitions 1 to 5, which has anaffinity to bind the O25b antigen with a Kd of less than 10⁻⁷ M,preferably less than 10⁻⁸ M.

7. Antibody according to any of definitions 1 to 6, which exhibits invitro bactericidal potency in a serum sample comprising live wild-typeMDR E. coli strains, and/or which stimulates uptake of live wild-typeMDR E. coli strains by phagocytic cells in vitro.

8. Antibody according to any of definitions 1 to 7, wherein the antibodybinds the same epitope as the antibody designated as 8D5-1G10 or8D10-C8.

9. Antibody according to any of definitions 1 to 8, wherein the antibodycomprises the same binding site as an antibody designated as 8D5-1G10 or8D10-C8.

10. Antibody according to any of definitions 1 to 9, wherein theantibody is derived from an antibody, which is characterized by avariable region, which is

-   obtainable from a host cell deposited under DSM 26763 and/or DSM    26762, or a functionally active variant thereof; or-   obtainable from a host cell deposited under DSM 28171 and/or DSM    28172, or a functionally active variant thereof.

11. Antibody according to definition 10, comprising A

-   a) a variable region of the antibody light chain produced by or    obtainable from a host cell deposited under DSM 26763; and/or-   b) a variable region of the antibody heavy chain produced by or    obtainable from a host cell deposited under DSM 26762;-   c) or a functionally active variant of (a) and/or (b)

or B

-   a) a variable region of the antibody light chain produced by or    obtainable from a host cell deposited under DSM 28171; and/or-   b) a variable region of the antibody heavy chain produced by or    obtainable from a host cell deposited under DSM 28172;-   c) or a functionally active variant of (a) and/or (b).

12. Antibody according to definition 10 or 11, wherein the functionallyactive variant comprises a CDR with an amino acid sequence having atleast 60% sequence identity.

13. Antibody according to any of definitions 10 to 12, wherein thefunctionally active variant differs from the parent antibody in at leastone point mutation in the amino acid sequence, preferably in the CDR,wherein the number of point mutations in each of the CDR amino acidsequences is either 0, 1, 2 or 3.

14. A plasmid comprising a nucleotide sequence A

-   encoding a variable region of the antibody light chain designated    8D5-1G10-LC comprised in a host cell deposited under DSM 26763;    and/or-   encoding a variable region of the antibody heavy chain designated    8D5-1G10-HC comprised in a host cell deposited under DSM 26762;

or B

-   encoding a variable region of the antibody light chain designated    8D10-C8-LC comprised in a host cell deposited under DSM 28171;    and/or-   encoding a variable region of the antibody heavy chain designated    8D10-C8-HC comprised in a host cell deposited under DSM 28172.

15. An expression cassette comprising a coding sequence to express alight chain and/or heavy chain of an antibody according to any of claims1 to 13, which expression cassette or coding sequence is derived from aplasmid according to definition 14.

16. Method of producing an antibody according to any of definitions 1 to13, wherein a host cell is transformed with a plasmid of claim 14 or theexpression cassette according to definition 15.

17. A host cell comprising a plasmid according to definition 14 or theexpression cassette according to definition 15.

18. The host cell according to definition 17, which is deposited under A

-   DSM 26763 and/or DSM 26762;

or B

-   DSM 28171 and/or DSM 28172.

19. Method of producing an antibody according to any of definitions 1 to13, wherein a host cell according to claim 17 or 18 is cultivated ormaintained under conditions to produce said antibody.

20. A method of identifying a candidate antibody comprising:

-   a) providing a sample containing an antibody or antibody-producing    cell; and-   b) assessing for binding of an antibody in or produced by the sample    with an epitope recognized by the antibody designated as 8D5-1G10 or    8D10-C8, wherein a positive reaction between the antibody and the    epitope identifies the antibody as candidate antibody.

21. A method of identifying a candidate antibody comprising:

-   a) providing a sample containing an antibody or antibody-producing    cell; and-   (b) assessing for binding of an antibody in or produced by the    sample with O25b antigen of a ST131-O25b:H4 strain and O25a antigen    of a non-MDR E. coli strain, wherein a specific positive reaction    between the antibody and the O25b antigen relative to the O25a    antigen identifies the antibody as candidate antibody.

22. A method of producing an antibody according to any of definitions 1to 13, comprising

-   a) providing a candidate antibody identified according to claim 20    or 21; and-   b) producing a monoclonal antibody, or a humanized or human form of    the candidate antibody, or a derivative thereof with the same    epitope binding specificity as the candidate antibody.

23. A method of producing an antibody according to any of definitions 1to 13, comprising

-   a) immunizing a non-human animal with an epitope recognized by the    antibody designated as 8D5-1G10 or 8D10-C8;-   b) forming immortalized cell lines from the isolated B-cells;-   c) screening the cell lines obtained in b) to identify a cell line    producing a monoclonal antibody that binds to the epitope; and-   d) producing the monoclonal antibody, or a humanized or human form    of the antibody, or a derivative thereof with the same epitope    binding specificity as the monoclonal antibody.

24. A method of producing an antibody according to any of definitions 1to 13, comprising

-   a) immunizing a non-human animal with O25b antigen of a    ST131-O25b:H4 strain and isolating B-cells producing antibodies;-   b) forming immortalized cell lines from the isolated B-cells;-   c) screening the cell lines to identify a cell line producing a    monoclonal antibody that preferentially binds to the O25b antigen    relative to the O25a antigen of E. coli; and-   d) producing the monoclonal antibody, or a humanized or human form    of the antibody, or a derivative thereof with the same epitope    binding specificity as the monoclonal antibody.

25. Antibody according to any of definitions 1 to 13, for use intreating a subject at risk of or suffering from a MDR E. coli infectioncomprising administering to the subject an effective amount of theantibody to limit the infection in the subject or to ameliorate adisease condition resulting from said infection, preferably fortreatment or prophylaxis of pyelonephritis, secondary bacteremia,sepsis, peritonitis, meningitis, and ventilator-associated pneumonia.

26. Antibody for use according to definition 25, for bactericidalkilling of MDR E. coli, preferably a ST131-O25b:H4 strain irrespectiveof capsular polysaccharide expressed by the strain.

27. Antibody for use according to definition 25 or 26, wherein theantibody is administered in a parenteral or mucosal formulation.

28. Pharmaceutical preparation of an antibody according to any ofdefinitions 1 to 13, preferably comprising a parenteral or mucosalformulation, optionally containing a pharmaceutically acceptable carrieror excipient.

29. Antibody according to any of definitions 1 to 13, for diagnostic useto determine E. coli infection in a subject caused by MDR strainsexpressing the LPS O25b, such as with upper and lower urinary tractinfections, including cystitis or urethritis, ascending or hematogenouspyelonephritis, especially in diabetic patients, as well as withbacteremia, sepsis, peritonitis, or intestinal colonization.

30. Antibody for use according to definition 29, wherein a systemicinfection with MDR E. coli in a subject is determined ex vivo bycontacting a sample of body fluid of said subject with the antibody,wherein a specific immune reaction of the antibody determines theinfection.

31. Antibody for use according to definition 29 or 30, wherein a sampleof body fluid is tested for the specific immune reaction, which sampleis selected from the group consisting of urine, blood, blood isolates orblood culture, aspirate, sputum, lavage fluid of intubated subjects andstool.

32. Antibody for use according to any of definitions 29 to 31, whereinthe serotype of E. coli is determined in vitro from a pure E. coliculture recovered from a clinical specimen.

33. Diagnostic preparation of an antibody according to any ofdefinitions 1 to 13, optionally containing the antibody with a labeland/or a further diagnostic reagent with a label and/or a solid phase toimmobilize at least one of the antibody and the diagnostic reagent.

34. Isolated epitope recognized by the antibody designated as 8D5-1G10or 8D10-C8.

35. An immunogen comprising:

-   a) an epitope according to definition 34;-   b) optionally further epitopes not natively associated with said    epitope of (a); and-   c) a carrier.

36. Immunogen according to definition 35, wherein said carrier is apharmaceutically acceptable carrier, preferably comprising buffer and/oradjuvant substances.

37. Immunogen according to definition 35 or 36, in a vaccineformulation, preferably for parenteral use.

38. Immunogen according to any of definitions 35 to 37, for use intreating a subject by administering an effective amount of saidimmunogen to protect the subject from an MDR E. coli infection, or toprevent a disease condition resulting from said infection.

39. Immunogen according to definition 38, for eliciting a protectiveimmune response.

40. Isolated nucleic acid encoding an antibody according to any ofdefinitions 1 to 13 or an epitope according to definition 35.

The foregoing description will be more fully understood with referenceto the following examples. Such examples are, however, merelyrepresentative of methods of practicing one or more embodiments of thepresent invention and should not be read as limiting the scope ofinvention.

EXAMPLES Example 1: O25b Specific Antibodies

We generated a non-encapsulated mutant of a representative ST131-O25b:H4strain 81009 (81009Δkps::kan, [Szijarto et al, FEMS Microbiol Lett,2012, 332:131-6]) by replacing the kps cluster (encoding capsularsynthesis) with a cassette encoding kanamycin resistance. Sublethaldoses of live or formaldehyde-inactivated cells of this mutant strainwere used to immunize mice 4 times at two-week intervals. Subsequently,serum samples obtained from the mice were analysed, and the spleen ofthe mouse showing the highest IgG titer against O25b antigen (in ELISA,immunoblotting, and surface staining) was used for hybridoma generation.Following sub-cloning, several hybridoma clones were selected, whichsecreted antibodies specific to purified O25b antigens as well as boundto the surface of live wild-type E. coli strains expressing O25bantigens. These mAbs were purified from hybridoma supernatants forfurther testing.

As depicted on FIG. 1 , all antibodies bound to several differentclinical isolates determined to be ST131-O25b:H4 strains irrespective ofthe capsular polysaccharide expressed (K5, K2, or unknown K types). Withrespect to binding to strains expressing the O25a antigen there were twotypes of mAbs identified. One group represented by mAb 8D5-1G10, did notbind to the surface of O25a strains, while the other group of mAbsrepresented by 8D10-C8 was cross-reactive to O25a strains. None of themAbs, however, could bind to any E. coli strains expressing unrelatedantigens, i.e. O2. (FIG. 1 ) or other O-types (not shown).

The specificity of mAbs was further confirmed by immunoblot analysisusing purified LPS (FIG. 2 ). The mAbs recognized the LPS molecules fromST131 strains containing the O25b antigen, however were different intheir cross-reactive potential to O25a LPS antigens. While mAb 8D5-1G10reacted exclusively to O25b antigen, mAb 8D10-C8 was cross-reactive toO25a. This observed cross-reactivity was compared to that exhibited bycommercial O25 typing serum (Statens Serum Institut, high titer E. coliO25 antiserum, #81369) routinely used for the detection of ST131:O25bstrains). The commercial rabbit serum showed clear preference towardsbinding to O25a antigens vs. O25b LPS. In contrast, murine mAb 8D10-C8reacted to O25b LPS molecules at least with the same intensity as to theO25a molecules. Subsequent quantitative analysis revealed that the ratioof binding intensity to O25b vs O25a is at least 10-fold higher in caseof mAb 8D10-C8 in comparison to the commercial typing serum.

The above data together suggest that there are two types ofO25b-specific mAbs, those, which are highly specific to O25b, and thosethat recognize an epitope shared by O25a and O25b antigens.Consequently, our data confirms that the structure of O25b indeeddiffers from that of classical O25 (i.e. O25a) antigen. The novelstructure of the O25b subunit was elucidated as described in Example 2.

The variable domains of the heavy (VH) and light (VL) chains ofO25b-specific mAbs were amplified from hybridoma clones by using RT-PCRwith degenerated heavy and light chain primers and sequenced. Sequenceswere analysed with BLAST for Ig database as well as with IMGT/V-QUEST,and CDR regions were defined according to Kabat nomenclature.

The variable light and heavy chain sequences of mAb 8D5-1G10 were clonedinto respective vectors, which were used to transform the E. coli hostcells deposited in DSMZ under accession numbers: DSM 26763 and DSM26762.

The variable light and heavy chain sequences of mAb 8D10-C8 were clonedinto respective vectors, which were used to transform the E. coli hostcells deposited in DSMZ under accession numbers: DSM 28171 and DSM28172.

Example 2: Structure Analysis of O25b Antigen

The LPS of E. coli ST131 was isolated by the hot phenol/water method andpurified by dialysis, proteinase K digest and ultracentrifugation. Theaverage yield of LPS preparations was 2.61% of dry bacterial mass. LPSwas analysed by SDS-PAGE, showing fractions consisting of coreoligosaccharide (OS) substituted with different numbers ofoligosaccharide repeating units (RU) as well as unsubstituted coreoligosaccharides. The O-specific polysaccharide (O-PS) and differentoligosaccharide components were released by mild acidic hydrolysis andisolated by gel filtration on Bio-Gel P-10. The fractions were analysedby sugar and methylation analyses, NMR spectroscopy and MALDI-TOF massspectrometry (MS).

The structure of the O-PS RU was determined with the use of a fractionconsisted of the core OS substituted with one single RU. Monosaccharideanalysis indicated the presence of Rha, Glc, Gal, Hep, and smalleramount of GlcN. Equimolar amounts of derivatives of terminal Rhap,terminal Glcp, 3,6-substituted Glcp, 3-substituted Rhap, with traceamounts 3-substituted GlcpN were identified and designated to the O-PSRU. Remaining partially methylated alditol acetates of 7-substitutedHepp, 6-substituted Glcp, 2-substituted Glcp, terminal Galp,3,6-substituted Glcp, and terminal Hepp constituted the coreoligosaccharide of K-12 type. Derivatives of 3,4-substituted Hepp,3,4,7-substituted Hepp, and Kdo could not be detected during standardmethylation and sugar analysis due to the substitution by P and PPEtnand presence of carboxyl group, respectively.

The structure of the RU of the O-specific PS of LPS ST131 was determinedwith the use of NMR spectroscopy. The complete assignment of the O-PS ¹Hand ¹³C resonances was achieved by combining the information obtainedfrom COSY, TOCSY and NOESY, as well as HSQC-DEPT, HMBC, and HSQC-TOCSYexperiments. The ¹H, ¹³C HSQC-DEPT spectrum contained signals for 13anomeric protons and carbons, and one Kdo spin system. These signalsderived from the core oligosaccharide as well as from the one RU of theO-specific PS. The high-field region contained one signal of CH₃ ofO-acetyl group, one signal of CH₃ of N-acetyl group, as well as twoupfield signals of CH₃ characteristic for the 6-deoxy sugars (Rha). Thespectra indicated a tetradecasaccharide structure of the investigatedoligosaccharide. Due to the high heterogeneity related to P, PP, andPEtn, the complete spin systems of eight sugars of the non-reducing endwere completely resolved with emphasis on the RU structure and itslinkage to the K-12 core OS.

The inter-residue connectivities between adjacent sugar residues wereobserved by NOESY and HMBC experiments. The HMBC spectra exhibitedcross-peaks between the anomeric proton and the carbon at the linkageposition and between the anomeric carbon and the proton at the linkageposition, which confirmed the sequence of sugar residues in thenon-reducing region of the polysaccharide.

Based on these measurements, the repeating unit of the O25b-specific PSwas determined (FIGS. 3 (a) ), which is a pentasaccharide with→3)-β-GlcpNAc (residue K) as a residue of RU substituting the firstresidue of the core OS: →7)-α-Hepp (residue L). Due to a possiblecontamination of the longer PS fractions, it was impossible to identifyposition of substitution of the first RU by subsequent RU of theO-specific chain. Moreover, without further detailed structural analysesof those fractions, the presence of GlcNAc in subsequent repeating unitsas α-anomer (what was previously reported for some of E. colilipopolysachcrides) could be ruled out.

Molecular weights of the core OS, core OS substituted with one RU, andfinally the O-PS RU was confirmed with the use of MALDI-TOF MS (data notshown). All spectra were interpreted on the basis of elucidated hereinstructure of the RU of LPS ST131 and previously identified glycoforms ofK-12 core OS (Duda et al. Microbiology. 2011 Jun;157(Pt 6):1750-60. doi:10.1099/mic.0.046912-0. Epub 2011 Mar 3; Muller-Loennies et al. J BiolChem. 2003 Sep 5;278(36):34090-101. Epub 2003 Jun 20). MALDI-MS analysisof low resolution spectra of fraction consisting of core OS substitutedwith shorter O-PS showed clusters of ions with following prevailingions: m/z 2797.2, m/z 3659.6, m/z 4522.0, and m/z 5383.6 attributed tocore OS (with P and PPEtn) substituted with 1, 2, 3, and 4 RUs,respectively. Average mass difference among these ions was 862.1 Da andmatched calculated average mass of the O-specific PS RU (861.8 Da,RU-H2O).

Taking into consideration the molecular weight of the RU and comparingMS results for other fractions, we have shown the presence of longercore OS glycoform consisting →7)-α-Hepp-(1→6)-α-Glcp disaccharide in theouter core region as a place of substitution with the first RU (FIGS. 3(a) ). It was shown that LPS ST131 consisted of two main core OSglycoforms. The type of glycoform is dependent on the presence orabsence of the O-specific PS. Prevailing glycoform of the unsubstitutedcore OS is truncated version of K-12 core oligosaccharide, which isdevoid of →7)-α-Hepp-(1→6)-α-Glcp disaccharide. Such disaccharide is thedifference between O-PS substituted core OS and non-substituted core OS.

Example 3: E.Coli O25b Specific Diagnostic Assay

The O25b-specific mAb 8D5-1G10 was bound to 1 µm diameter latex beads(Polysciences) by following instructions of the manufacturer.Latex-coupled beads were tested for their potential to agglutinatedifferent E. coli strains. A loopful of bacteria (approx. 10⁸ cfu) wasmixed with 10 µl of 1% suspension of mAb-coupled latex beads in PBS. Asdepicted on FIG. 4 E. coli strains expressing O25b antigens showed astrong agglutination pattern after gentle agitation for a few seconds.On the contrary, E. coli strains expressing O25a or O2 antigens did notagglutinate with the same reagent. Therefore, this putative diagnosticreagent is considered to be more specific than the currently used stateof the art agglutination reagent (i.e., polyclonal rabbit serum againstO25) used for the detection of O25b (and O25a)- positive E. coli.

Furthermore, as commercial anti-O25 serum is recommended to be used withheat killed (i.e., lysed) E. coli cells in agglutination assays, wetested, whether O25b mAbs - either purified or coupled to latex beads -would have a higher sensitivity, i.e., whether they are able toagglutinate live E. coli cells in the presence of intact capsularpolysaccharides. A large panel of O25b clinical isolates were tested andresults with some representative strains are presented in Table I.Improvement in sensitivity was found in two respects: i) representativestrains #1 and #2 were agglutinable with both free or bead-coupledO25b-specific mAbs in a non-heat killed (i.e. live bacteria directlytaken from an agar plate) form, while agglutination with the commercialO25 rabbit serum required prior heat-lysis of the bacteria, ii)representative strains #3 and #4 were not able to be agglutinated withthe commercial serum even in a heat killed form, whereas the samelysates gave positive result with the purified mAb 8D5-1G10.Importantly, when the same antibody was coupled to latex beads,agglutination developed even with native bacterial cells. These resultsindicate a superior sensitivity of the bead-coupled mAbs in anagglutination assays, which is corroborated by the fact that all O25bpositive E. coli strains tested so far gave a positive agglutinationwith this reagent even without any prior treatment (i.e. withoutproducing a heat-killed lysate). Moreover, using this reagent as adiagnostic tool for the detection of O25b-expressing bacteria has theadvantage over PCR based technique that it only gives a positive resultwith bacteria in fact expressing the target antigen. For instance,representative strain #5 in Table I was PCR positive for the O25bspecific gene routinely used in diagnostics, however, was negative inany agglutination assays. This strain has been proven to exhibit a roughLPS phenotype (no O-antigens expressed), therefore, the PCR result couldbe considered as false positive. Avoiding such false positivity is ofgreat importance, when such assays are used as companion diagnostics,i.e., to select patients infected with O25b expressing E. coli strainthat could benefit from O25b specific therapeutic approaches.

The potential of detecting free O25b LPS molecules by the latexbead-coupled mAbs was also tested. Different amounts of highly purifiedO25b LPS in the range of 1-1000 ng were incubated with 10 µl of 1% beadsuspension in PBS. As depicted on FIG. 5 , a dose dependentagglutination pattern was seen: the best results were obtained with 100ng of free LPS, agglutination was still detectable with 1000 or 10 ng,while was undetectable with 1 ng of free O25b LPS.

TABLE I Comparison of agglutination results obtained with various O25bstrains using commercial O25 typing serum and O25b specific mAb 8D5-1G10O25b strain rfb_(O25b) PCR Agglutination by O25 typing serum(commercial) of heat-killed lysates Agglutination by O25b mAbs ofAgglutination by 8D5-1G10 coupled beads of live (non heat-killed)bacteria Lysates (heat- killed) Live cells (non heat-killed)#1 + + + + + #2 + + + + + #3 + - - + + #4 + - - + + #5 + - - - -

Example 4: Antibacterial Effect of O25b Specific mAbs

The potential protective effect of O25b-specific mAbs (with or withoutcross-reactivity to O25a) was tested in a lethal murine bacteremiamodel. Groups of 5 mice received 100 µg of purified 8D5-1G10 or 8D10-C8,intraperitoneally. 24 h later mice were challenged intravenously by alethal dose (previously determined in a pilot experiment) of E. colistrain 81009 (2×10⁸ CFU/mouse) expressing the O25b antigen. Lethality ofmice was monitored daily for 3 weeks. FIG. 6 shows combined results of 2independent experiments with similar outcome. While 90% of the mice mockimmunized with PBS succumbed to infection, both mAbs tested providedstatistically (Logrank test) significant increase in survival over the3-week post-infection period monitored.

In order to corroborate this in vivo data, bactericidal effect ofpurified mAbs was also tested in vitro. 2 ml of a mid-log culture of E.coli strain 81009 was washed twice in PBS and re-suspended to a finalconcentration of 5×10⁵ CFU/ml. 10 µl of this bacterial suspension waspre-incubated for 15 minutes at 4° C. with 4 µg of the respective mAbsdiluted in 40 µl RPMI-1640 buffer supplemented with 3% human albumin.Subsequently, 50 µl of pooled human serum (previously adsorbed with E.coli strain 81009) was added to the reaction and incubated at 37° for 1,2 and 3 hrs. The final CFU and antibody concentrations in the reactionwere 5×10⁴ CFU/ml and 40 µg/ml respectively in a total volume of 100 µl.10 µl aliquots were plated onto TSB plates for colony counting at thespecified time points.

As depicted on FIG. 7 , both mAbs tested were able to significantlydecrease the CFU over the 3 hours study period. In contrast, thebacteria mixed with an irrelevant mAb or no antibodies showed constantgrowth in this medium. In case complement was inactivated in the serumsamples (by 30 min. incubation at 56° C.), no bacterial killing wasobserved by any mAbs (data not shown). These results prove that bothO25b-specific mAbs can trigger complement mediated bactericidal effect.

1-40. (canceled)
 41. A method of determining an infection with multidrug resistant (MDR) E. coli strains in a subject, comprising contactinga sample of body fluid of the subject with an antibody that specificallybinds to an O25b antigen having a repeating unit of the structure of:

wherein a specific immune reaction of the antibody determines the E.coli infection.
 42. The method of claim 41, wherein the antibodycomprises: (i) the variable region of the antibody light chain producedby a host cell deposited under DSM 26763 and (ii) the variable region ofthe antibody heavy chain produced by a host cell deposited under DSM26762; or (i) the variable region of the antibody light chain producedby a host cell deposited under DSM 28171 and (ii) the variable region ofthe antibody heavy chain produced by a host cell deposited under DSM28172.
 43. The method of claim 41, wherein the antibody furthercomprises a label or reporter molecule.
 44. The method of claim 43,wherein the label or reporter molecule is selected from the groupconsisting of organic molecules, enzyme labels, radioactive labels,colored labels, fluorescent labels, chromogenic labels, luminescentlabels, haptens, digoxigenin, biotin, metal complexes, metals, colloidalgold and mixtures thereof.
 45. The method of claim 41, furthercomprising contacting the sample with a diagnostic reagent specificallyrecognizing the antibody or an immune complex of the antibody with theO25b antigen.
 46. The method of claim 41, wherein the antibody isimmobilized on a solid phase.
 47. The method of claim 46, wherein thesolid phase comprises latex beads or gold particles.
 48. The method ofclaim 41, wherein the sample is selected from the group consisting ofurine, blood, blood isolates or blood culture, aspirate, sputum, lavagefluid, and stool.
 49. The method of claims 41, wherein the infection isa urinary tract infection, an ascending or hematogenous pyelonephritis,a bacteremia, sepsis, peritonitis, or intestinal colonization.
 50. Themethod of claim 41, further comprising administering a therapeutic totreat the infection.
 51. The method of claim 50, wherein the therapeuticcomprises a monoclonal antibody comprising (i) the variable region ofthe antibody light chain produced by a host cell deposited under DSM26763 and (ii) the variable region of the antibody heavy chain producedby a host cell deposited under DSM 26762; or (i) the variable region ofthe antibody light chain produced by a host cell deposited under DSM28171 and (ii) the variable region of the antibody heavy chain producedby a host cell deposited under DSM
 28172. 52. A kit comprising: (a) anisolated monoclonal antibody that specifically binds to an O25b antigencomprising (i) the variable region of the antibody light chain producedby a host cell deposited under DSM 26763 and (ii) the variable region ofthe antibody heavy chain produced by a host cell deposited under DSM26762; or (i) the variable region of the antibody light chain producedby a host cell deposited under DSM 28171 and (ii) the variable region ofthe antibody heavy chain produced by a host cell deposited under DSM28172; and (b) instructions for use.
 53. The kit of claim 52, whereinthe antibody further comprises a label.
 54. The kit of claim 52, furthercomprising a diagnostic reagent specifically recognizing the antibody oran immune complex of the antibody with the O25b antigen.
 55. The kit ofclaim 54, further comprising a solid phase to immobilize at least one ofthe antibody and the diagnostic reagent.
 56. The kit of claim 55,wherein the solid phase comprises latex beads or gold particles.
 57. Thekit of claim 52, wherein the antibody is in the lyophilized form. 58.The kit of claim 57, wherein the kit further comprises a medium toreconstitute the lyophilizate.
 59. An antibody or an antigen bindingfragment thereof that binds specifically to an O25b antigen having arepeating unit of the structure of.